Covalently linked antigen-antibody conjugates

ABSTRACT

Herein is reported a conjugate comprising an antigen and an antibody that specifically binds to the antigen wherein a covalent bond is formed between the antigen and an amino acid residue in the CDR2 of the antibody, an antibody comprising a cysteine residue at amino acid position 53 (according to Kabat) in the heavy chain CDR2 and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2013/064100 having and international filing date of Jul. 4, 2013,the entire contents of which are incorporated herein by reference, andwhich claims benefit under 35 U.S.C. § 119 to European PatentApplication No. 12174947.7, filed Jul. 4, 2012.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web. Said ASCII copy, created on Dec. 16, 2014, is namedP31087_US_C_SeqList.txt., and is 107,313 bytes in size.

Herein are reported complexes comprising an antigen and an antibodywhereby the components of the complex are covalently linked to eachother via a single bond. Also reported are methods for producing thecovalent complexes and uses thereof.

BACKGROUND OF THE INVENTION

Major bottlenecks for therapeutic application of polypeptides are theirlimited solubility, in vivo stability, short serum half-life and fastclearance from the bloodstream.

Different approaches are reported to address these drawbacks. However,none of these technologies provides for a robust and universal platformthat enables pharmacokinetic (PK) modulation without encounteringimmunogenicity risks or potential loss of biological activity.

One approach to improve PK/stability and biophysical behavior oftherapeutic polypeptides is to fuse them to entities which stabilizedthe polypeptide, keep it in solution, and extend its half-life. Examplesof such entities are human serum albumin or human immunoglobulinFc-regions. This approach is applicable to many linear polypeptides thatare composed of naturally occurring amino acid residues and thattolerate modifications at either their C- or N-terminus without losingtheir biological activity. Polypeptides that are cyclic, stapled,contain non-natural amino acid residues, or additional modificationscannot be recombinantly produced as fusion polypeptides. However, suchpolypeptides may be the desired choice for therapeutic applicationsbecause they are frequently superior to ‘normal’ linear peptides interms of protease stability, activity and specificity.

One approach to improve PK/stability and biophysical behavior oftherapeutic polypeptides, which can also be applied to those that arecyclic, stapled, or contain non-natural structures, is the chemical orenzymatic conjugation to polymers, for example by PEGylation orHESylation. However, such modifications frequently lead to significantreduction of the biological activity of the polypeptide and can undercertain circumstances be the reason for safety or toxicity problems.

A major disadvantage of most existing chemical coupling technologies forstabilization or PK modulation of therapeutic polypeptides is theircomplexity. Beside the chemical coupling step the methods result in manycases in a mixture of polypeptide derivatives that are connected to thePK-modulating entity with uncertain stoichiometries and/or at undefinedpositions. Additionally currently used polypeptidemodification-technologies often result in strongly reduced or evencomplete loss of biological activity of the therapeutic polypeptide. Inaddition, it is difficult to predict pharmacological properties and/orpossible degradation routes of the chemical conjugates.

U.S. Pat. No. 5,804,371 reports hapten-labeled peptides and their use inan immunological method of detection. A digoxigenin-labeled peptide(Bradykinin) and its application to chemiluminoenzyme immunoassay ofBradykinin in inflamed tissues are reported by Decarie A., et al.(Peptides 15 (1994) 511-518).

In WO 2004/065569 multi-functional antibodies are reported.

SUMMARY OF THE INVENTION

It has been found that by the covalent coupling of a haptenylatedcompound to an anti-hapten antibody a stabilization and PK-propertyimprovement of the compound can be achieved.

One aspect as reported herein is a conjugate comprising an antigen andan antibody that specifically binds to the antigen characterized by acovalent bond between the antigen and an amino acid residue in the CDR2of the antibody, whereby the CDR2 is determined according to Kabat.

It has been found that any antigen can be used in the conjugates andmethods as reported herein upon derivatization with a universal linkerwhich comprises the functional residue for the formation of the covalentbond between the antigen and an amino acid residue in the CDR2 of theantibody. The location of the functional group in the universal linkerhas the advantage that it is not necessary to re-engineer the synthesisand the position of the functional group in the CDR2 of the antibody.

One aspect as reported herein is a conjugate comprising a haptenylatedcompound and an antibody that specifically binds to the hapten of thehaptenylated compound (anti-hapten antibody) characterized by a covalentbond between the haptenylated compound and an amino acid residue in theCDR2 of the antibody, whereby the CDR2 is determined according to Kabat.

In one embodiment of all aspects the CDR2 is the heavy chain CDR2.

In one embodiment of all aspects the haptenylated compound comprises ahapten, an optional linker, and a payload.

In one embodiment of all aspects the covalent bond is between the linkerof the haptenylated compound and an amino acid residue in the CDR2 ofthe antibody.

In one embodiment of all aspects the covalent bond is between afunctional group in the haptenylated compound and the amino acid residuein the CDR2 of the antibody.

In one embodiment the functional group is in the linker of thehaptenylated compound.

In one embodiment of all aspects as reported herein the covalent bond isbetween a cysteine residue in the heavy chain CDR2 of the antibody and afunctional group in the antigen or a functional group in thehaptenylated compound.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52, or position 52a, or position 52b, orposition 52c, or position 52d, or position 53 according to the heavychain variable domain numbering of Kabat.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52a, or position 52b, or position 52c, orposition 53 according to the heavy chain variable domain numbering ofKabat.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52b or at position 53 according to the heavychain variable domain numbering of Kabat.

In one embodiment of all aspects the covalent bond is a disulfide bond.

In one embodiment of all aspects the antibody is a bispecific antibodycomprising a first binding specificity to a first antigen or a firsthapten and a second binding specificity to a second antigen or a secondhapten, wherein the first antigen/hapten is different from the secondantigen/hapten.

In one embodiment the antibody comprises a first binding specificity toa first hapten and a second binding specificity to a second hapten.

In one embodiment the antibody comprises a first binding specificity toa hapten and a second binding specificity to an (non-hapten) antigen.

In one embodiment the (non-hapten) antigen is a cell surface antigen. Inone embodiment the cell surface antigen is a tumor associated antigen.

In one embodiment the bispecific antibody is a full length antibody. Inone embodiment one heavy chain of the bispecific antibody comprises ahole mutation and the respective other chain comprises a knob mutation.

In one embodiment of all aspects the payload is selected from a bindingmoiety, a labeling moiety, and a biologically active moiety.

In one embodiment of all aspects the antibody is a full length antibody.

In one embodiment of all aspects the antibody is a humanized or a humanantibody.

In one embodiment the constant region of the antibody is of the IgG1subclass or of the IgG4 subclass.

In one embodiment the antibody has a constant region of the IgG1subclass with an alanine at position 234 and 235 and with a glycine atposition 329 with numbering according to the EU index of Kabat.

In one embodiment the antibody has a constant region of the IgG4 classwith a proline at position 228, a glutamic acid at position 235 and aglycine at position 329 with numbering according to the EU index ofKabat.

In one embodiment of all aspects as reported herein the antibody is afragment of an antibody. In one embodiment the fragment is a Fab or a(Fab)₂.

In one embodiment of all aspects the conjugate comprises exactly onecovalent bond per heavy chain CDR2.

In one embodiment of all aspects as reported herein the antigen or thehaptenylated compound comprises a reactive group that can form acovalent bond with the thiol group of the cysteine residue in the CDR2of the antibody. In one embodiment the reactive group is a thiol, or amaleimide, or a haloacetyl.

In one embodiment of all aspects as reported herein the covalent bond isa disulfide bond and it is formed without the addition of redox activeagents.

In one embodiment of all aspects the conjugate comprises a therapeuticor detectable moiety. In one embodiment the therapeutic or detectablemoiety is covalently conjugated to the antigen or the hapten.

In one embodiment the antigen or hapten is conjugated to a polypeptideconsisting of 5 to 60 amino acid residues. In one embodiment thepolypeptide comprises 10 to 50 amino acid residues. In one embodimentthe polypeptide comprises 12 to 40 amino acid residues. In oneembodiment the polypeptide comprises 12 to 30 amino acids residues.

In one embodiment the antigen or hapten is conjugated to a detectablelabel.

In one embodiment the antigen or the hapten is conjugated to thepolypeptide, or to the detectable label, or to the payload via a linker.In one embodiment the linker is a non-peptidic linker.

One aspect as reported herein is an antibody that has in the lightand/or the heavy chain a cysteine residue in the CDR2 whereby the CDRsare determined according to Kabat.

In one embodiment the cysteine residue is in the heavy chain CDR2.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52, or position 52a, or position 52b, orposition 52c, or position 52d, or position 53 according to the heavychain variable domain numbering of Kabat.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52a, or position 52b, or position 52c, orposition 53 according to the heavy chain variable domain numbering ofKabat.

In one embodiment the cysteine residue in the heavy chain CDR2 of theantibody is at position 52b or at position 53 according to the heavychain variable domain numbering of Kabat.

In one embodiment the antibody has in exactly one heavy chain variabledomain a cysteine residue at position 52b or position 53.

In one embodiment the hapten is biotin, or theophylline, or digoxigenin,or carborane, or fluorescein.

In one embodiment the antibody is a humanized or human antibody.

In one embodiment the antibody is a full length antibody, or a Fab, or ascFv, or a scFv conjugated to an Fc-region.

In one embodiment the cysteine forms a disulfide bond with an isolatedcysteine residue or an isolated homocysteine residue

One aspect as reported herein is an immunoconjugate comprising theconjugate as reported herein and a cytotoxic agent.

One aspect as reported herein is a pharmaceutical formulation comprisingthe conjugate as reported herein and a pharmaceutically acceptablecarrier.

The conjugate as reported herein for use as a medicament.

The conjugate as reported herein for the treatment of cancer.

The conjugate as reported herein for the treatment of diabetes.

The conjugate as reported herein for the treatment of adiposities.

The conjugate as reported herein for the treatment of an inflammatorydisease.

The conjugate as reported herein for the treatment of a metabolicdisease.

The conjugate as reported herein for the treatment of a viral disease.

One aspect as reported herein is the use of a conjugate as reportedherein in the manufacture of a medicament.

One aspect as reported herein is the use of a conjugate as reportedherein as diagnostic agent.

One aspect as reported herein is the use of a conjugate as reportedherein comprising a therapeutic polypeptide to increase the stability ofthe therapeutic polypeptide.

One aspect as reported herein is the use of a conjugate as reportedherein comprising a therapeutic polypeptide to increase the activity ofthe therapeutic polypeptide.

One aspect as reported herein is the use of a conjugate as reportedherein comprising a therapeutic polypeptide to increase the in vivohalf-life of the therapeutic polypeptide.

One aspect as reported herein is the use of a conjugate as reportedherein in the treatment of a disease.

One aspect as reported herein is a method of treating an individualhaving a disease comprising administering to the individual an effectiveamount of a conjugate as reported herein.

One aspect as reported herein is a method of treating a disease in anindividual comprising administering to the individual an effectiveamount of the conjugate as reported herein.

In one embodiment the disease is cancer.

In one embodiment the disease is diabetes.

In one embodiment the disease is adipositas.

One aspect as reported herein is a method of producing a conjugate asreported herein comprising the combination of an antibody comprising afirst reactive group and an antigen that has a second reactive groupwhereby the alpha carbon atom of the amino acid residue that bears thefirst reactive group is about 10 to 11 Angstrom apart from the atom ofthe antigen to which the linker is fused.

One aspect as reported herein is a method of producing a conjugate asreported herein comprising the steps of

-   -   combining in solution an antibody that specifically binds to a        hapten and comprises a reactive group at one amino acid residue        in the CDR2 with a haptenylated compound comprising a reactive        group, wherein the haptenylated compound comprises a payload,        such as a peptide consisting of 5 to 60 amino acids or a        detectable label, and    -   recovering of the conjugate from the solution.

One aspect as reported herein is a method for producing an antibody forthe formation of a conjugate as reported herein, comprising the step of

-   -   cultivating a cell comprising a nucleic acid encoding the        antibody, and    -   recovering the antibody from the cell or the cultivation medium,

wherein in the antibody the residue in the heavy chain CDR2 is mutatedto cysteine that has in the X-ray structure of the non-covalent complexof the antibody and its antigen or a haptenylated compound a distance of10 to 11 Angstrom between the alpha-carbon atom of the amino acidresidue in the antibody CDR2 and the atom of the hapten to which thelinker is connected or to an antigen atom between which the covalentbond is to be formed.

One aspect as reported herein is a method for identifying a position inan antibody CDR2 that can be mutated to cysteine for the formation of acovalent bond between the residue in the antibody CDR2 and the boundantigen or haptenylated compound comprising the step of

-   -   providing a crystal structure of the non-covalent complex of the        antibody and the antigen or haptenylated compound, whereby the        antigen or haptenylated compound comprises a linker sequence,        and    -   identifying an amino acid residue in the CDR2 of the antibody        and in the linker sequence with a distance of 10 to 11 Angstrom        between the alpha-carbon atoms of the amino acid residue in the        antibody CDR2 and the atom in the antigen to which the linker is        fused,

wherein the identified position is the position in an antibody CDR2 thatcan be mutated to cysteine for the formation of a covalent bond betweenthe residue in the antibody CDR2 and the bound antigen or haptenylatedcompound.

One aspect as reported herein is a bispecific antibody for targeteddelivery of a haptenylated compound to a target cell, wherein thebispecific antibody comprises a first binding site that specificallybinds to the haptenylated compound and a second binding specificity thatspecifically binds to a cell surface marker of the cell.

DESCRIPTION OF THE FIGURES

FIG. 1: Procedure for digoxigenylation (conjugation of digoxigenin to)of peptides (FIG. 1A). Examples of a digoxigenylated label (fluorophoreDig-Cy5; FIG. 1B) and of a digoxigenylated polypeptide (PYY-derivative(DIG-PYY); FIG. 1C).

FIG. 2: Scheme of a complex of a monospecific bivalent anti-digoxigeninantibody and a digoxigenin-Cy5 conjugate (FIG. 2A) and of a complex of amonospecific bivalent anti-digoxigenin antibody and adigoxigenin-polypeptide conjugate (FIG. 2B). Scheme of a complex of abispecific tetravalent anti-digoxigenin antibody and adigoxigenin-polypeptide conjugate (FIG. 2C).

FIG. 3: Size exclusion chromatogram (recorded at 280 nm) of a complexcomprising an anti-digoxigenin antibody and digoxigenin which isconjugated to a peptide (DIG-PYY) showing a single peak of a complex ofdefined size.

FIG. 4: A: Structure model of an anti-digoxigenin Fab (left) showingthat digoxigenin (encircled) is captured in a deep pocket which isformed by the CDRs of the VH and VL regions. B: Structure model of ananti-biotin Fab (right) showing that biocytinamid (encircled) iscaptured in a deep pocket which is formed by the CDRs of the VH and VLregions.

FIG. 5: Comparison of the binding of recombinant humanized anti-biotinantibodies with and without introduced VH53C mutation. Bindingproperties were analyzed by surface plasmon resonance (SPR) technologyusing a BIAcore T100 or BIAcore 3000 instrument. a) humanizedanti-biotin antibody. Binding of biotinylated siRNA to humanizedanti-biotin antibody, KD=624 pM; b) humanized Cys53 mutated anti-biotinantibody. Binding of biotinylated siRNA, KD=643 pM; siRNAconcentrations: 0.14, 0.41, 1.23, 3.70, 11.1, 33.3, and 100 nM;anti-biotin antibody concentration: 2 nM; Sensor Chip CM3; binding ofantibody via anti-human IgG Fc antibody

ka (1/Ms) kd (1/s) KD (M) humanized anti-biotin 2.2 * 10⁷ 0.01 6.4 *10⁻¹⁰ antibody VH53C humanized anti-biotin 2.0 * 10⁷ 0.01 6.2 * 10⁻¹⁰antibody

FIG. 6: Introduction of SH functionalities in the hapten as well as inthe antibody at appropriate positions allow the antibody and the haptento form a covalent bond in between resulting in a conjugate.

FIG. 7: Scheme of SDS-PAGE self-fluorescence band pattern (withoutfurther staining of the SDS-PAGE gel):

-   -   A: If no covalent bond is formed between the antibody and the        hapten-fluorophore conjugate both under reducing or non-reducing        conditions one self-fluorescent band at the molecular weight of        free hapten-fluorophore conjugate can be detected.    -   B: If a covalent bond is formed between the antibody and the        hapten-fluorophore conjugate under non-reducing conditions one        self-fluorescent band at the combined molecular weight of the        antibody and the hapten-fluorophore conjugate can be detected.        Under reducing conditions the disulfide bridges in the conjugate        of the antibody and the hapten-fluorophore conjugate        (haptenylated compound) are cleaved and one self-fluorescent        band at the molecular weight of free hapten-fluorophore        conjugate can be detected.

FIG. 8: Conjugate formation of hapten-binding Cys-mutated antibodieswith hapten-Cys-fluorescent label conjugates (haptenylated compound) inthe presence of redox active agents: oxidation agent (glutathionedisulfide, GSSG) and reducing agent (dithioerythritol, DTE): Antibodycomplexation and subsequent covalent linkage at defined positions isdetected by fluorescence signals in SDS PAGE analyses. Non-reducing(upper images) and reducing (lower images) SDS-PAGE analyses wereperformed as described in Example 11. Covalently antibody linked haptensare detectable as larger sized protein bound signals at the appropriatepositions under non-reduced conditions. These signals detach fromprotein upon reduction and are visible as small entities under reducingconditions.

-   -   Left: fluorescence image    -   Right: Coomassie blue staining    -   Series 1: anti-digoxigenin antibody with 52bC mutation    -   Series 2: anti-digoxigenin antibody with wild-type residue at        position 52b    -   (A) covalent coupling with 3 mM DTE and 10 mM GSSG;    -   (B) covalent coupling with 0.3 mM DTE and 1 mM GSSG;    -   (C) covalent coupling with 0.03 mM DTE and 0.1 mM GSSG.

FIG. 9: Complex formation of hapten-binding Cys mutated antibodies withhapten-Cys-fluorescent label conjugates in the presence solely of anoxidation agent (glutathione disulfide, GSSG) but in the absence ofreducing agents or in the absence of both: Antibody complexation andsubsequent covalent linkage at defined positions is detected byfluorescence signals in SDS PAGE analyses. Non-reducing (upper images)and reducing (lower images) SDS-PAGE analyses were performed asdescribed in Example 12. Covalently antibody linked haptens aredetectable as larger sized protein bound signals at the appropriatepositions under non-reduced conditions. These signals detach fromprotein upon reduction and are visible as small entities under reducingconditions.

-   -   Left: fluorescence image    -   Right: Coomassie blue staining    -   Series 1: anti-digoxigenin antibody with 52bC mutation    -   Series 2: anti-digoxigenin antibody with wild-type residue at        position 52b    -   (A) no additives    -   (B) covalent coupling with 1 mM GSSG;    -   (C) covalent coupling with 0.1 mM GSSG.

FIG. 11: Structure of DIG-3-cme-eda-Cy5

FIG. 12: Structure of DIG-maleiimid-Cy5

FIG. 13: Structure of DIG-eda-Cys-Cy5

FIG. 14: Structure of DIG-Ahx-Cys-Cy5

FIG. 15: Structure of DIG-Cys-MR121

FIG. 16: Structure of Ac-PYY(PEG3-Dig)

FIG. 17: Structure of Ac-PYY(PEG3-Cys-4Abu-Dig)

FIG. 18: Structure of PEG3-PYY(PEG3-Cys-4Abu-Dig)

FIG. 19: Structure of Dy636-eda-Btn

FIG. 20: Structure of Dy636-Ser-Btn

FIG. 21: Structure of Dy636-Cys-Btn

FIG. 22: Structure of Cy5-Cys-Btn

FIG. 23: Structure of Cy5-Ser-Btn

FIG. 24: Structure of Ac-PYY(PEG2-Btn)

FIG. 25: Structure of Ac-PYY-PEG3-Cys-β-Ala-Btn)

FIG. 26: Structure of Ac-PYY-PEG3-Ser-PEG2-Btn)

FIG. 27: Structure of Ac-PYY-PEG3-Cys-PEG2-Btn

FIG. 28: Structure of Ac-PYY(PEG3-Cys-4-Abu-5-Fluo)

FIG. 29: Structure of Ac-PYY(PEG3-Cys-PEG2-5-Fluo)

FIG. 30: Scheme for the generation of Ac-PYY(PEG2-Btn)

FIG. 31: Scheme for the generation of Ac-PYY(PEG3-Cys-β-Ala-Btn)

FIG. 32: Scheme for the generation of Ac-PYY(PEG3-Cys-4-Abu-Dig)

FIG. 33: X-ray structure of murine anti-biotin antibody in complex withbiocytinamid. Amino acid residues that are interacting with biocytinamidare shown in a stick representation.

FIG. 34A and FIG. 34B:

Results of the in vivo blood PK study with covalent conjugates andnon-covalent complexes compared to non-complexed antigen/hapten; therelative remaining fluorescence intensity (%, solid marks) ofCy5-mediated fluorescence of Biotin-Cy5 non-covalent complexes (FIG.34A) and covalent (SS-bridged) conjugates (FIG. 34B), as well as ofnon-complexed Biotin-Ser-Cy5 (asterix) is shown; the fluorescence signalat time point t=0.08 h was set to 100%; additionally, the relativeremaining amount of human IgG in the mouse serum samples is shown (openmarks); IgG serum concentration (mg/ml) at t=0.08 h was set to 100%.

FIG. 35: Western blot of the determination of the amount ofdigoxigenylated PYY polypeptide in the serum of mice.

FIG. 36: Analysis of affinity-driven complexation of haptenylatedcompounds with anti-hapten antibodies.

-   -   Antibody complexation and subsequent covalent linkage at defined        positions is directed by fluorescence signals in SDS PAGE        analyses, which were carried out as described in Example 19.    -   Left: fluorescent image with non-reduced (left side of gel) and        reduced (right side of gel) samples.    -   Right: Coomassie blue staining.    -   1: humanized anti-digoxigenin antibody+biotin-Cys-Cy5    -   2: humanized anti-digoxigenin antibody VH52bC+biotin-Cys-Cy5    -   3: humanized anti-biotin antibody+biotin-Cys-Cy5    -   4: humanized anti-biotin antibody VH53C+biotin-Cys-Cy5    -   The white arrows mark the excess (uncoupled) biotin-Cys-Cy5,        which is significantly higher when anti-digoxigenin antibody        VH52bC is used, because the conjugation reaction is not affinity        driven in this case.

FIG. 37: Cysteine positions and disulfide patterns within the Fabregion, required to form a Dig-binding antibody with additional cysteineat position 52b for hapten-mediated site-directed directed covalentpayload coupling. (A) Cysteines and disulfide pattern in VH and CH1domains, and in VL and CL domains that are required to form functionalFab fragments. (B) Cysteines and disulfide pattern in VH and CH1domains, and in VL and CL domains that are required to form functionalFab fragments with additional cysteine at position 52b forhapten-mediated site-directed directed covalent payload coupling. (C&D)Potential to form incorrect disulfide bonds within the VH domain of theVH52b variant which would result in misfolded nonfunctional antibodies.E) Example for a potential incorrect interdomain disulfide bond withinthe Fv region of the VH52b variant, which would result in misfoldednonfunctional antibodies.

FIG. 38: Cysteine positions and disulfide patterns required to form aDig-binding disulfide-stabilized single-chain Fv with additionalcysteine at position 52b for hapten-mediated site-directed directedcovalent payload coupling. (A) Cysteines in VH and VL domains that arerequired to form functional scFvs, dsscFvs and 52b mutated dsscFvs. (B)correct pattern of disulfide bonds that must be formed to generatefunctional scFvs, dsscFvs and 52b mutated dsscFvs. (C) Potential to formincorrect disulfide bonds which would result in misfolded nonfunctionalscFvs. (D) Potential to form incorrect disulfide bonds which wouldresult in misfolded nonfunctional dsscFvs. (E) Potential to formincorrect disulfide bonds which would result in misfolded nonfunctional52b mutated dsscFvs.

FIG. 39: Composition of a LeY-Dig bispecific antibody derivative asdelivery vehicle for covalently coupled payloads.

FIG. 40: Expression and Purification of bispecific anti-hapten antibodyderivatives for targeted delivery of covalently coupled payloads.

-   -   (A) For Western blot analyses, cell culture supernatants were        subjected to SDS PAGE (NuPAGE 4-12% Bis-Tris Gel (1.0 mm×12        well) (Invitrogen; Cat. No. NP0322) and proteins were        subsequently transferred to Immobilon Transfer Membranes        (Immobilon-P) (Millipore; Cat. No. IPVH07850), PVDF with pore        Size: 0.45 μm. Antibody derivatives were detected by Anti-Human        Kappa Light Chain)-Alkaline Phosphatase antibody produced in        goat, (affinity purified), Sigma (Cat. No. A3813) at a 1:1000        dilution, and Anti-Human IgG (Fc specific)-Alkaline Phosphatase        antibody produced in goat, Sigma (Cat. No. A9544) at a 1:1000        dilution. The substrate BCIP/NBT-Blue Liquid Substrate (Sigma        Cat. No. B3804 was applied for the development of the Western        blot. Lane 1—molecular weight marker; Lane 2 & 3-control        antibody with unmodified heavy-chain; Lane 4 LeY-Dig(52bC)        bispecific antibody with extended H-chain.    -   (B) SDS-PAGE analyses (NuPAGE 4-12% Bis-Tris Gel [Invitrogen]        and subsequent staining with Coomassie brilliant blue        demonstrates purity of protein preparations and visualizes        polypeptide chains related to the IgG with the apparent        molecular sizes that correspond to their calculated molecular        weights. Lane 1—molecular weight marker; Lane 2-LeY-Dig(52bC)        bispecific antibody with extended H-chain reduced, lane        3-LeY-Dig(52bC) bispecific antibody with extended heavy-chain        non-reduced;    -   (C) Size exclusion chromatography (Superdex 200) demonstrates        homogeneity and lack of aggregates in the protein preparations        of the LeY-Dig(52bC) bispecific antibody derivative after        Protein A purification.

FIG. 41: Relative remaining fluorescence intensity (%) of Cy5-mediatedfluorescence of Dig-Cy5 non-covalent complexes and covalent(disulfide-bridged) conjugates, as well as of non-complexed Dig-Cy5; thefluorescence signal at time point t=0.08 h was set to 100%;additionally, the relative remaining amount of human IgG in the mouseserum samples is shown; IgG serum concentration (mg/ml) at t=0.08 h wasset to 100%.

FIG. 42: Pharmacokinetics under in vivo-like conditions of Cy5-mediatedfluorescence of Biotin-Cy5 of non-covalent complexes and of covalent(disulfide-bridged) conjugates, as well as of non-complexed Biotin-Cy5,determined by non-invasive eye imaging; solid diamond: biotin-Cy5, solidsquare biotin-Cy5+anti-biotin antibody (complex); triangle:Cy5-Biotin-anti-biotin antibody conjugate.

FIG. 43: a) Composition, structure and molecular weight ofTheophylline-Cys-Cy5; b) Size exclusion chromatography demonstratespurity and homogeneity of purified theophylline-binding antibodyvariants; peak #2 shows the purified product, lack of peak #1 indicatesthat such preparations are free of aggregates; c) formation of covalentcomplexes between theophylline-binding antibodies andTheophylline-Cys-Cy5 as demonstrated by non-reducing (left lanes) andreducing (right lanes) SDS PAGE; Cy5 appears coupled to the H-chainunder non-reducing conditions only in samples that containedTheophylline-Cys-Cy5 and cys-mutated antibody, these covalent conjugatesdisintegrate upon reduction (right lanes); Lanes 1: Molecular weightmarker; 2-4 non-reducing—2: anti-Theophylline antibody (withoutcys-mutation)+Theophylline-Cys-Cy5 (complex); 3: anti-Theophyllineantibody-cys_55+Theophylline-Cys-Cy5 (conjugate); 4: anti-Theophyllineantibody-cys_54+Theophylline-Cys-Cy5 (conjugate); 5-7 reducing—5:anti-Theophylline antibody (without cys-mutation)+Theophylline-Cys-Cy5(complex); 6: anti-Theophylline antibody-cys_55+Theophylline-Cys-Cy5(conjugate); 7: anti-Theophylline antibody-cys_54+Theophylline-Cys-Cy5(conjugate).

FIG. 44: Formation of covalent complexes between biotin-bindingantibodies and Biotin-Cys-Cy5 is demonstrated by non-reducing andreducing SDS PAGE; the coupling reaction was performed in murine serumat 37° C. for 1 hr. Cy5 appears coupled to the H-chain undernon-reducing conditions only in samples that contained Biotin-Cys-Cy5and cys-mutated antibody; these covalent conjugates disintegrate uponreduction (right lanes); lanes 1: Molecular weight marker; 2-3non-reducing—2: anti-Biotin antibody (without cysmutation)+Biotin-Cys-Cy5 (complex); 3: anti-Biotinantibody-cys+Biotin-Cys-Cy5 (conjugate); 4-5 reducing—5: anti-Biotinantibody (without cys mutation)+Biotin-Cys-Cy5 (complex); 6: anti-Biotinantibody-cys+Biotin-Cys-Cy5 (conjugate)

FIG. 45: In vivo pharmacokinetics of Cy5-mediated fluorescence ofBiotin-Cy5 of non-covalent complexes and of covalent (disulfide-bridged)conjugates, as well as of non-complexed Biotin-Cy5, determined bynon-invasive eye imaging; solid diamond: biotin-Cy5, solid circle:biotin-Cy5 administered 24 hours after administration of anti-biotinantibody (in vivo complex formation); solid square: biotin-Cys-Cy5administered 24 hours after administration of anti-biotin antibody-cys(in vivo conjugate formation).

FIG. 46: The protein structure of murine anti-Biotinantibody-Fab-fragment was determined in complex with biocytinamid: thecomplexed hapten is positioned in close proximity to a negativelycharged cluster of amino acids; biotin which—as hapten—is derivatizedfor payload coupling at its carboxyl group binds with good efficacy asthere is no charge repulsion at this position (due to the lack of theCOOH group); in contrast, free (normal) biotin cannot bind efficient tothe antibody because its carboxyl group would be in close proximity tothis negative charge cluster, and hence becomes repulsed.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “amino acid” denotes the group of carboxy α-amino acids, eitheroccurring naturally, i.e. which directly or in form of a precursor canbe encoded by a nucleic acid, or occurring non-naturally. The individualnaturally occurring amino acids are encoded by nucleic acids consistingof three nucleotides, so called codons or base-triplets. Each amino acidis encoded by at least one codon. This is known as “degeneration of thegenetic code”. The term “amino acid” as used within this applicationdenotes the naturally occurring carboxy α-amino acids comprising alanine(three letter code: ala, one letter code: A), arginine (arg, R),asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C),glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine(his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K),methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine(ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y),and valine (val, V). Examples of non-naturally occurring amino acidsinclude, but are not limited to, Aad (alpha-Aminoadipic acid), Abu(Aminobutyric acid), Ach (alpha-aminocyclohexane-carboxylic acid), Acp(alpha-aminocyclopentane-carboxylic acid), Acpc(1-Aminocyclopropane-1-carboxylic acid), Aib (alpha-aminoisobutyricacid), Aic (2-Aminoindane-2-carboxylic acid; also called 2-2-Aic),1-1-Aic (1-aminoindane-1-carboxylic acid), (2-aminoindane-2-carboxylicacid), Allylglycine (AllylGly), Alloisoleucine (allo-Ile), Asu(alpha-Aminosuberic acid, 2-Aminooctanedioc acid), Bip(4-phenyl-phenylalanine-carboxylic acid), BnHP((2S,4R)-4-Hydroxyproline), Cha (beta-cyclohexylalanine), Cit(Citrulline), Cyclohexylglycine (Chg), Cyclopentylalanine,beta-Cyclopropyl alanine, Dab (1,4-Diaminobutyric acid), Dap(1,3-Diaminopropionic acid), p (3,3-diphenylalanine-carboxylic acid),3,3-Diphenylalanine, Di-n-propylglycine (Dpg), 2-Furylalanine,Homocyclohexylalanine (HoCha), Homocitrulline (HoCit), Homocycloleucine,Homoleucin (HoLeu), Homoarginine (HoArg), Homoserine (HoSer),Hydroxyproline, Lys(Ac), (1) Nal (1-Naphtyl Alanine), (2) Nal (2-NaphtylAlanine), 4-MeO-Apc(1-amino-4-(4-methoxyphenyl)-cyclohexane-1-carboxylic acid), Nor-leucine(Nle), Nva (Norvaline), Omathine, 3-Pal(alpha-amino-3-pyridylalanine-carboxylic acid), 4-Pal(alpha-amino-4-pyridylalanine-carboxylic acid), 3,4,5,F3-Phe(3,4,5-Trifluoro-phenylalanine), 2,3,4,5,6,F5-Phe(2,3,4,5,6-Pentafluoro-phenylalanine), Pqa(4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (CAS889958-08-1)), Pyridylalanine, Quinolylalanine, Sarcosine (Sar),Thiazolylalanine, Thienylalanine, Tic(alpha-amino-1,2,3,4,tetrahydroisoquinoline-3-carboxylic acid), Tic(OH),Tle (tertbutylGlycine), and Tyr(Me).

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% sequence identity with the native sequence polypeptide.In one embodiment the variant has about 80% or more sequence identitywith the native sequence polypeptide. In one embodiment the variant hasabout 90% or more sequence identity with the native sequencepolypeptide. In one embodiment the variant has about 95% or moresequence identity with the native sequence polypeptide. In oneembodiment the variant has about 98% or more sequence identity with thenative sequence polypeptide. The amino acid sequence variants possesssubstitutions, deletions, and/or insertions at certain positions withinthe amino acid sequence of the native amino acid sequence Amino acidsare designated by the conventional names, one-letter and three-lettercodes.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “antibody fragment” denotes a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “biotin”, short “BI”, denotes5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoicacid. Biotin is also known as vitamin H or coenzyme R.

The term “bispecific antibodies” denotes antibodies which have twodifferent (antigen/hapten) binding specificities. In one embodimentbispecific antibodies as reported herein are specific for two differentantigens, i.e. a hapten and a non-hapten antigen.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153,Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “digoxigenin”, short “DIG”, denotes3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydro-cyclopenta[a]-phenanthren-17-yl]-2H-furan-5-one(CAS number 1672-46-4). Digoxigenin (DIG) is a steroid found exclusivelyin the flowers and leaves of the plants Digitalis purpurea, Digitalisorientalis and Digitalis lanata (foxgloves) (Polya, G., Biochemicaltargets of plant bioactive compounds, CRC Press, New York (2003) p.847).

The term “effector functions” denotes those biological activitiesattributable to the Fc-region of an antibody, which vary with theantibody class. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation.

The term “effective amount” of an agent, e.g., a pharmaceuticalformulation, denotes an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic or prophylacticresult.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

The term “Fc-region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc-regions andvariant Fc-regions. In one embodiment, a human IgG heavy chain Fc-regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc-regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc-region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

The term “fluorescein”, short “FLUO”, denotes6-hydroxy-9-(2-carboxyphenyl)-(3H)-xanthen-3-on, alternatively2-(6-hydroxy-3-oxo-(3H)-xanthen-9-yl)-benzoic acid. Fluorescein is alsoknown as resorcinolphthalein, C.I. 45350, solvent yellow 94, D & Cyellow no. 7, angiofluor, Japan yellow 201, or soap yellow.

The term “framework”, short “FR”, denotes heavy and light chain variabledomain amino acid residues other than hypervariable region (HVR)residues. The FR of a variable domain generally consists of four FRdomains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequencesgenerally appear in the following sequence in VH (or VL):FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “free cysteine amino acid” denotes a cysteine amino acidresidue which has been engineered into a parent antibody, has a thiolfunctional group (SH), and is not paired as an intramolecular disulfidebridge. Nevertheless, a free cysteine amino acid can be pair asintramolecular disulfide bridge, e.g. with glutathione.

The term “full length antibody” denotes an antibody having a structuresubstantially similar to a native antibody structure or having heavychains that contain an Fc-region as defined herein. Native IgGantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light chains and two identical heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3). Similarly, from N- to C-terminus, each light chain has a variableregion (VL), also called a variable light domain or a light chainvariable domain, followed by a constant light (CL) domain. The lightchain of an antibody may be assigned to one of two types, called kappa(κ) and lambda (λ), based on the amino acid sequence of its constantdomain.

A “full length antibody” is an antibody comprising a VL and VH domain,as well as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or anamino acid sequence variant thereof. The full length antibody may haveone or more “effector functions” which refer to those biologicalactivities attributable to the Fc constant region (a native sequenceFc-region or amino acid sequence variant Fc-region) of an antibody.Examples of antibody effector functions include C1q binding; complementdependent cytotoxicity; Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation ofcell surface receptors such as B-cell receptor and BCR.

The term “hapten” denotes a small molecule that can elicit an immuneresponse only when attached to a large carrier such as a protein.Exemplary haptens are aniline, o-, m-, and p-aminobenzoic acid, quinone,histamine-succinyl-glycine (HSG), hydralazine, halothane, indium-DTPA,fluorescein, biotin, digoxigenin, theophylline and dinitrophenol. In oneembodiment the hapten is biotin or digoxigenin or theophylline orcarborane.

The term “a hapten that is conjugated to” or “haptenylated compound”denotes to a hapten which is covalently linked to a further moiety suchas a polypeptide or a label. Activated hapten derivatives are often usedas starting materials for the formation of such conjugates. In oneembodiment the hapten is digoxigenin and it is conjugated (in oneembodiment via its 3-hydroxy group) to the moiety via a linker. In oneembodiment the linker comprises a) one or more (in one embodiment threeto six) methylene-carboxy-methyl groups (—CH₂—C(O)—), and/or b) from 1to 10 (in one embodiment from 1 to 5) amino acid residues (in oneembodiment selected from glycine, serine, glutamate, β-alanine,γ-aminobutyric acid, ε-aminocaproic acid or lysine), and/or c) one ormore (in one embodiment one or two) compounds having the structuralformula NH₂—[(CH₂)_(n)O]_(x)CH₂—CH₂—COOH in which n is 2 or 3 and x is 1to 10, in one embodiment 1 to 7. The last element results (at leastpartly) in a linker (part) of the formula—NH—[(CH₂)_(n)O]_(x)CH₂—CH₂—C(O)—. One example of such a compound ise.g. 12-amino-4,7,10-trioxadodecanoic acid (results in a TEG(triethylenglycol) linker). In one embodiment the linker furthercomprises a maleimido group. The linker has a stabilizing andsolubilizing effect since it contains charges or/and can form hydrogenbridges. In addition it can sterically facilitate the binding of theanti-hapten antibody to the hapten-conjugated polypeptide. In oneembodiment the linker is located at a side chain of an amino acid of thepolypeptide (e.g. conjugated to a lysine or cysteine side chain via anamino or thiol group). In one embodiment the linker is located at theamino terminus or at the carboxy terminus of the polypeptide. Theposition of the linker on the polypeptide is typically chosen at aregion where the biological activity of the polypeptide is not affected.Therefore the attachment position of the linker depends on the nature ofthe polypeptide and the relevant structure elements which areresponsible for the biological activity. The biological activity of thepolypeptide to which the hapten attached can be tested in an in vitroassay.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanERs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the ERscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”), and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3).

HVRs herein include

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987)        901-917);    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat, E. A. et al., Sequences of Proteins of Immunological        Interest, 5th ed. Public Health Service, National Institutes of        Health, Bethesda, Md. (1991), NIH Publication 91-3242);    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “monospecific antibody” denotes an antibody that has one ormore binding sites each of which has the same binding specificity, i.e.binds to the same antigen or hapten.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native orwild-type sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild-type, or modified forms ofan antibody. The parent antibody binds specifically to a hapten. Aparent antibody may be directed additionally also against a targetantigen of interest, e.g. a biologically important polypeptide.Antibodies directed against non-polypeptide antigens are alsocontemplated.

The term “payload” denotes any molecule or combination of moleculeswhose activity it is desired to be delivered (in)to and/or localize at acell. Payloads include, but are not limited to labels, cytotoxins (e.g.Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and the like),enzymes, growth factors, transcription factors, drugs, radionuclides,ligands, antibodies, liposomes, nanoparticles, viral particles,cytokines, and the like.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamylamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, chlorophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitroureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhóne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-II; 35 topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic agent may, for instance, be a small molecule or anantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The anti-angiogenic factor is in oneembodiment an antibody that binds to Vascular Endothelial Growth Factor(VEGF).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-a and -P;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-p; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-a andTGF-p; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-a, -P, and -y;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF);interleukins (ILs) such as IL-I, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-IO, IL-II, IL-12; a tumor necrosis factor such asTNF-α or TNF-P; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “fMLP” denotes the tripeptide consisting of N-formylmethionine,leucine and phenylalanine. In one embodiment the effector moiety is fMLPor a derivative thereof.

The term “prodrug” refers to a precursor or derivative form of apharmaceutically active substance that is less cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into the more active parent form. See, e.g.,Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, Vol. 14, 615th Meeting Belfast (1986) pp. 375-382 andStella, et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery”, Directed Drug Delivery, Borchardt, et al., (eds.), pp.247-267, Humana Press (1985). The prodrugs that can be used as effectormoiety include, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, b-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described herein.

The term “cytotoxic moiety” refers to a substance that inhibits orprevents a cellular function and/or causes cell death or destruction.Cytotoxic agents include, but are not limited to, radioactive isotopes(e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents); growth inhibitory agents;enzymes and fragments thereof such as nucleolytic enzymes; antibiotics;toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof; and the various antitumor or anticancer agentsdisclosed herein.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

A “polypeptide” is a polymer consisting of amino acids joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 20 amino acid residues may be referred to as “peptides”,whereas molecules consisting of two or more polypeptides or comprisingone polypeptide of more than 100 amino acid residues may be referred toas “proteins”. A polypeptide may also comprise non-amino acidcomponents, such as carbohydrate groups, metal ions, or carboxylic acidesters. The non-amino acid components may be added by the cell, in whichthe polypeptide is expressed, and may vary with the type of cell.Polypeptides are defined herein in terms of their amino acid backbonestructure or the nucleic acid encoding the same. Additions such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

All polypeptide sequences are written according to the generallyaccepted convention whereby the alpha-N-terminal amino acid residue ison the left and the alpha-C-terminal amino acid residue is on the right.As used herein, the term “N-terminus” refers to the free alpha-aminogroup of an amino acid in a polypeptide, and the term “C-terminus”refers to the free a-carboxylic acid terminus of an amino acid in apolypeptide. A polypeptide which is N-terminated with a group refers toa polypeptide bearing a group on the alpha-amino nitrogen of theN-terminal amino acid residue. An amino acid which is N-terminated witha group refers to an amino acid bearing a group on the alpha-aminonitrogen.

Unless indicated otherwise by a “D” prefix, e.g., D-Ala or N-Me-D-Ile,or written in lower case format, e.g., a, i, 1, (D versions of Ala, Ile,Leu), the stereochemistry of the alpha-carbon of the amino acids andaminoacyl residues in polypeptides described in this specification andthe appended claims is the natural or “L” configuration. TheCahn-Ingold-Prelog “R” and “S” designations are used to specify thestereochemistry of chiral centers in certain acyl substituents at theN-terminus of the polypeptides. The designation “R,S” is meant toindicate a racemic mixture of the two enantiomeric forms. Thisnomenclature follows that described in Cahn, R. S., et al., Angew. Chem.Int. Ed. Engl. 5 (1966) 385-415.

The term “single-chain Fv”, short “scFv”, denotes an antibody fragmentthat comprise the VH and VL domains of antibody, wherein these domainsare present in a single polypeptide chain. In one embodiment, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Plueckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore (Eds),Springer-Verlag, New York, pp. 269-315 (1994).

The term “theophylline”, short “THEO”, denotes1,3-dimethyl-7H-purine-2,6-dione. Theophylline is also known asdimethylxanthine.

The term “treatment” (and grammatical variations thereof such as “treat”or “treating”) denotes a clinical intervention in an attempt to alterthe natural course of the individual being treated, and can be performedeither for prophylaxis or during the course of clinical pathology.Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or to slow theprogression of a disease.

The term “x-valent”, e.g. “mono-valent” or “bi-valent” or “tri-valent”or “tetra-valent”, denotes the presence of a specified number of bindingsites, i.e. “x”, in an antibody molecule. As such, the terms “bivalent”,“tetravalent”, and “hexavalent” denote the presence of two binding site,four binding sites, and six binding sites, respectively, in an antibodymolecule. The bispecific antibodies as reported herein are at least“bivalent” and may be “trivalent” or “multivalent” (e.g. “tetravalent”or “hexavalent”). In one embodiment the bispecific antibody as reportedherein is bivalent, trivalent, or tetravalent. In one embodiment thebispecific antibody is bivalent. In one embodiment the bispecificantibody is trivalent. In one embodiment the bispecific antibody istetravalent.

In certain aspects and embodiments the antibodies as reported hereinhave two or more binding sites and are bispecific. That is, theantibodies may be bispecific even in cases where there are more than twobinding sites (i.e. that the antibody is trivalent or multivalent). Theterm bispecific antibodies includes, for example, multivalent singlechain antibodies, diabodies and triabodies, as well as antibodies havingthe constant domain structure of full length antibodies to which furtherantigen-binding sites (e.g., single chain Fv, a VH domain and/or a VLdomain, Fab, or (Fab)2,) are linked via one or more peptide-linkers. Theantibodies can be full length from a single species, or be chimerized orhumanized. For an antibody with more than two antigen binding sites,some binding sites may be identical, so long as the protein has bindingsites for two different antigens. That is, whereas a first binding siteis specific for a hapten, a second binding site is specific for anon-hapten antigen, and vice versa.

The term “variable region” denotes the domain of an antibody heavy orlight chain that is involved in binding the antibody to its antigen. Thevariable domains of the heavy chain and light chain (VH and VL,respectively) of a native antibody generally have similar structures,with each domain comprising four conserved framework regions (FRs) andthree hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. KubyImmunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) Asingle VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993)880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term “vector” denotes a nucleic acid molecule capable of propagatinganother nucleic acid to which it is linked. The term includes the vectoras a self-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. Certain vectors are capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “expression vectors”.

II. Conjugates as Reported Herein

In one aspect the invention is based on the finding that a covalentconjugate comprising an antigen and an antibody that specifically bindsto the antigen can be obtained by the formation of a covalent bondbetween a properly placed functional group in the antigen and an aminoacid residue comprising a functional group in a variable domain of theantibody, especially in the CDR2 of the antibody, whereby the CDR2 isdetermined according to heavy chain variable domain numbering accordingto Kabat.

In certain embodiments the antigen is a hapten. In one embodiment thehapten is biotin, or digoxigenin, or fluorescein, or theophylline, orcarborane.

In one embodiment the antigen is a haptenylated compound. In oneembodiment the haptenylated compound is a conjugate comprising a hapten,a linker and a payload. In one embodiment the hapten is biotin, ordigoxigenin, or fluorescein, or theophylline, or carborane.

In certain embodiments the functional group may contain electrondeficient double bonds or a thiol. In one embodiment the functionalgroup is a maleimide or a cysteine.

In certain aspects of the invention antibodies are provided thatspecifically bind to digoxigenin.

In certain aspects of the invention antibodies are provided thatspecifically bind to biotin.

In certain aspects of the invention antibodies are provided thatspecifically bind to theophylline.

As the conjugates as reported herein can be used a therapeutic agent inhumans also humanized variants of the above antibodies againstdigoxigenin, biotin, and theophylline are provided herein.

Covalent conjugates of a hapten-polypeptide conjugate (haptenylatedcompound) and an anti-hapten antibody may confer benign biophysicalbehavior and improved PK properties to the polypeptide. Furthermore, incase a bispecific antibody is used, the conjugates can be used to targetthe polypeptide to cells which display the antigen that is recognized bythe second binding specificity of the bispecific antibody. Suchconjugates are composed of one anti-hapten binding specificity and one(non-hapten) antigen binding specificity. The stoichiometric ratio ofantibody to hapten-polypeptide conjugate depends on the format of thebispecific antibody and can be 1:1, 1:2, 2:1, 2:2, 2:4 and 4:2(antibody:hapten-polypeptide).

It is desired that the polypeptide retains good biological activitydespite being conjugated the hapten, as well as being conjugated to theantibody. It is also desired (in case of bispecific targeting modules)that the cell surface target binding site of the bispecific antibodyretains its binding specificity and affinity in the presence of thecovalently conjugated haptenylated compound.

The reactive group in the antigen (hapten) may be any reactive group,such as e.g. a maleimide, e.g. N-ethyl maleimide (NEM), a iodoacetamide,a pyridyl disulfide, or other reactive conjugation partner (see e.g.Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals, Molecular Probes, Inc.; Brinkley, 1992, BioconjugateChem. 3:2; Garman, 1997, Non-Radioactive Labeling: A Practical Approach,Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson,G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp.40-55 and 643-671).

The reactive group on the antibody is limited to those that can beselectively, i.e. position specifically, generated. Therefore, it islimited to the side chain groups of the amino acid residues cysteine,serine, asparagine, glutamine, tyrosine, lysine, arginine, asparticacid, and glutamic acid.

For the formation of a covalent conjugate between the antibody and theantigen/hapten both compounds have to be modified by the introduction ofa reactive group. Upon binding of the antigen/hapten by the antibody thetwo reactive groups are brought in close proximity allowing theformation of a covalent bond. In one embodiment the modification is theintroduction of a thiol functionality in each of the compounds. In oneembodiment the thiol compound is a cysteine residue.

The position to be mutated must simultaneously meet two requirements:(i) the coupling positions should be in proximity to the binding regionto utilize the antigen/hapten positioning effect for directed coupling,and (ii) the mutation and coupling position must be positioned in amanner that antigen/hapten binding by itself is not affected. Theserequirements for finding a suitable position are de facto‘contradicting’ each other because requirement (i) is best served by aposition close to the binding site, while requirement (ii) is mostsafely achieved by positions that are distant from the binding site.

Despite these virtually excluding requirements, positions wereidentified that can be mutated without affecting hapten positioning, andwhich nevertheless simultaneously allow directed covalent coupling.

The first position is located at position VH52b or at position VH53,respectively, according to the Kabat numbering of the heavy chainvariable domain. If the antibody has a short VH CDR2, which does nothave intermittent residues, such as 52a, 52c, 52c, and 52d, the positionis 53 (numbering and alignment according to the numbering scheme andrules of Kabat for the antibody heavy chain variable domain). If theantibody has a long VH CDR2 comprising residues 52a and 52b, andoptionally further residues as 52c and 52d, etc. the position is 52b(numbering and alignment according to the numbering scheme and rules ofKabat for the antibody heavy chain variable domain).

The second position is located at position VH28 according to the Kabatnumbering.

For example, in the anti-digoxigenin antibody structure, the hapten isbound in a deep pocket formed by hydrophobic residues. A fluorescentdigoxigenin-Cy5 conjugate was used in this crystallographic study,wherein the fluorophore as well as the linker between digoxigenin andCy5 were not visible in the structure due to a high flexibility andresulting disorder in the crystal. However, the linker and Cy5 areattached to 032 of digoxigenin which points into the direction of theCDR2 of the heavy chain. The distance between 032 of digoxigenin to theCα of the amino acid residue in position 52b according to Kabat is about10.5 Å.

It has been found that the positions are “universal” position, i.e. theposition is applicable to any (anti-hapten) antibody and, thus, it isnot required to start from scratch every time a new (anti-hapten)antibody has to be modified by providing the crystal structure anddetermining the appropriate position that enables hapten-positionedcovalent coupling.

The mutation VH52bC or VH53C, respectively, according to the numberingscheme of Kabat could unexpectedly be used for each hapten-bindingantibody analyzed. Even though the antibodies and structures of theirbinding pockets are quite diverse, it has been shown that theVH52bC/VH53C mutation can be used for covalent attachment ofantigens/haptens to antibodies that bind digoxigenin, biotin,fluorescein, as well as theophylline. Thus, it has further been foundthat the change of the amino acid residue at position VH52b/VH53according to the numbering according to Kabat, which residue is insidethe heavy chain CDR2 of the antibody, to cysteine is applicable to other(anti-hapten) antibodies, without further need of structural design orknowledge of specific antibody structures, and without interference inthe binding properties inherent to the variable domains of the antibody.

The antibodies modified as reported herein retain the hapten (antigen)binding capability of their parent (i.e. wild-type) antibodycounterparts. Thus, the engineered antibody is capable of binding, inone embodiment it is capable of specifically binding, to haptens(antigens).

The terms “binding specificity” or “an antibody that binds to” denotethat the molecule comprising the binding specificity or an antibody canform a complex with a further molecule in a specific manner. The bindingcan be detected in an in vitro assay, such as in a plasmon resonanceassay (BIAcore, GE-Healthcare Uppsala, Sweden). The affinity of thecomplex formation is defined by the terms ka (rate constant for theassociation of the compounds to form the complex), k_(D) (dissociationconstant, dissociation of the complex), and K_(D) (k_(D)/ka). Binding orspecifically binding means a binding affinity (K_(D)) of about 10⁻⁸ M orless, in one embodiment of about 10⁻⁸ M to about 10⁻¹³ M, in oneembodiment of about 10⁻⁹ M to about 10⁻¹³ M. Thus, an antibody thatbinds to a hapten to form a complex as reported herein specificallybinds to the hapten with a binding affinity (K_(D)) of about 10⁻⁸ mol/lor less, in one embodiment of about 10⁻⁸ mol/l to about 10⁻¹³ mol/1, inone embodiment of about 10⁻⁹ mol/l to 10⁻¹³ mol/1.

It has been found that the formation of a covalent bond between acysteine-modified antibody and a cysteine-modified hapten-polypeptideconjugate bearing the cysteine residue in the linker between the haptenand the polypeptide takes place upon binding of the antibody to thehapten without the requirement of the addition of reducing and/oroxidizing agents if the formed bond is a disulfide bond. Thus, thedisulfide bridge between the two compounds is formed spontaneously uponformation of the non-covalent complex. Therefore, a method for theformation of a covalent complex as reported herein simply requires themixing of the two compounds. The only pre-requisite for the formation ofthe disulfide bond is a proper orientation of the two compounds withrespect to each other.

The engineered antibodies as reported herein may be site-specificallyand efficiently coupled with a hapten comprising a reactive group.

Replacement of the amino acid residue at position VH52b and VH53,respectively, (according to the Kabat numbering scheme) with a Cysresidue resulted in antibody derivatives with heavy chain variableregion sequences that are listed in SEQ ID NO: 20 and 28 foranti-digoxigenin antibody-VH52bC, in SEQ ID NO: 84 and 92 foranti-theophylline antibody-VH53C, in SEQ ID NO: 52 and 60 foranti-biotin antibody-VH53C, and in SEQ ID NO: 108 for anti-fluoresceinantibody-VH52bC.

Replacement of the heavy chain variable domain amino acid residue atposition VH28 (according to the Kabat numbering scheme) with a Cysresidue resulted in antibody derivatives with heavy chain variableregion sequences that are listed in SEQ ID NO: 116, 124, 132, 140, 148,156, and 164, respectively.

One aspect as reported herein is an anti-digoxigenin antibody that is ahumanized antibody.

In one aspect as reported herein an anti-digoxigenin antibody isprovided comprising at least one, two, three, four, five, or six CDRsselected from (a) heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 09 or 25, (b) heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO: 10 or 26, (c) heavy chain CDR3 comprising theamino acid sequence of SEQ ID NO: 11 or 27, (d) light chain CDR1comprising the amino acid sequence of SEQ ID NO: 13 or 29, (e) lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or 30,and (f) light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 15 or 31.

One aspect as reported herein is an anti-biotin antibody that is ahumanized antibody.

In one aspect as reported herein an anti-biotin antibody is providedcomprising at least one, two, three, four, five, or six CDRs selectedfrom (a) heavy chain CDR1 comprising the amino acid sequence of SEQ IDNO: 41 or 57, (b) heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 42 or 58, (c) heavy chain CDR3 comprising the amino acidsequence of SEQ ID NO: 43 or 59, (d) light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 45 or 61, (e) light chain CDR2comprising the amino acid sequence of SEQ ID NO: 46 or 62, and (f) lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 47 or 64.

One aspect as reported herein is an anti-fluorescein antibody comprisingat least one, two, three, four, five, or six CDRs selected from (a)heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 105 or113, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ IDNO: 106 or 114, (c) heavy chain CDR3 comprising the amino acid sequenceof SEQ ID NO: 107 or 115, (d) light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 109 or 117, (e) light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 110 or 118, and (f) light chain CDR3comprising the amino acid sequence of SEQ ID NO: 111 or 119.

One aspect as reported herein is an anti-theophylline antibody. In oneembodiment the anti-theophylline is a humanized antibody.

In one embodiment an anti-theophylline antibody is provided comprisingat least one, two, three, four, five, or six CDRs selected from (a)heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 73 or89, (b) heavy chain CDR2 comprising the amino acid sequence of SEQ IDNO: 74 or 90, (c) heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 75 or 91, (d) light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 77 or 93, (e) light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 78 or 94, and (f) light chain CDR3comprising the amino acid sequence of SEQ ID NO: 79 or 95.

One aspect as reported herein is an anti-digoxigenin antibody comprising(a) a VH domain comprising at least one, at least two, or all three VHCDR sequences selected from (i) heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 09 or 25, (ii) heavy chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 10 or 26, and (iii) heavy chainCDR3 comprising an amino acid sequence of SEQ ID NO: 11 or 27, and (b) aVL domain comprising at least one, at least two, or all three VL CDRsequences selected from (i) light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 13 or 29, (ii) light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 14 or 30, and (c) light chain CDR3comprising the amino acid sequence of SEQ ID NO: 15 or 31.

One aspect as reported herein is an anti-biotin antibody comprising (a)a VH domain comprising at least one, at least two, or all three VH CDRsequences selected from (i) heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO: 41 or 57, (ii) heavy chain CDR2 comprising theamino acid sequence of SEQ ID NO: 42 or 58, and (iii) heavy chain CDR3comprising an amino acid sequence of SEQ ID NO: 43 or 59, and (b) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (i) light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 45 or 61, (ii) light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 46 or 6242, and (c) light chain CDR3comprising the amino acid sequence of SEQ ID NO: 47 or 63.

One aspect as reported herein is an anti-fluorescein antibody comprising(a) a VH domain comprising at least one, at least two, or all three VHCDR sequences selected from (i) heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 105 or 113, (ii) heavy chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 106 or 114, and (iii) heavy chainCDR3 comprising an amino acid sequence of SEQ ID NO: 107 or 115, and (b)a VL domain comprising at least one, at least two, or all three VL CDRsequences selected from (i) light chain CDR1 comprising the amino acidsequence of SEQ ID NO: 109 or 117, (ii) light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 110 or 118, and (c) light chain CDR3comprising the amino acid sequence of SEQ ID NO: 111 or 119.

One aspect as reported herein is an anti-theophylline antibodycomprising (a) a VH domain comprising at least one, at least two, or allthree VH CDR sequences selected from (i) heavy chain CDR1 comprising theamino acid sequence of SEQ ID NO: 73 or 89, (ii) heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 74 or 90, and (iii)heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 75 or91, and (b) a VL domain comprising at least one, at least two, or allthree VL CDR sequences selected from (i) light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 77 or 93, (ii) light chain CDR2comprising the amino acid sequence of SEQ ID NO: 78 or 94, and (c) lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 79 or 95.

In any of the above aspects and embodiment, the anti-digoxigeninantibody, and/or the anti-biotin antibody, and/or the anti-theophyllineantibody is humanized.

In one embodiment, an anti-digoxigenin antibody comprises CDRs as in anyof the above aspects and/or embodiments, and further comprises anacceptor human framework, e.g. a human immunoglobulin framework or ahuman consensus framework.

In one embodiment, an anti-biotin antibody comprises CDRs as in any ofthe above aspects and/or embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, an anti-theophylline antibody comprises CDRs as inany of the above aspects and/or embodiments, and further comprises anacceptor human framework, e.g. a human immunoglobulin framework or ahuman consensus framework.

Another aspect as reported herein is an anti-digoxigenin antibody thatcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 04 or 12 or 20 or 28.In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-digoxigenin antibody comprising thatsequence retains the ability to bind to digoxigenin. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 01 or 09 or 17 or 25. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (i.e., in the FRs). Optionally, the anti-digoxigeninantibody comprises the VH sequence in SEQ ID NO: 01 or 09 or 17 or 25,including post-translational modifications of that sequence.

Another aspect as reported herein is an anti-digoxigenin antibody thatcomprises a light chain variable domain (VL) having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 08 or 16 or 24 or 32. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-digoxigenin antibody comprising thatsequence retains the ability to bind to digoxigenin. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 08 or 16 or 24 or 32. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the CDRs (i.e., in the FRs). Optionally, theanti-digoxigenin antibody comprises the VL sequence in SEQ ID NO: 08 or16 or 24 or 32, including post-translational modifications of thatsequence.

Another aspect as reported herein is an anti-biotin antibody thatcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 36 or 44 or 52 or 60.In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-biotin antibody comprising thatsequence retains the ability to bind to biotin. In certain embodiments,a total of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 36 or 44 or 52 or 60. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theCDRs (i.e., in the FRs). Optionally, the anti-biotin antibody comprisesthe VH sequence in SEQ ID NO: 36 or 44 or 52 or 60, includingpost-translational modifications of that sequence.

Another aspect as reported herein is an anti-fluorescein antibody thatcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 108 or 116. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-fluorescein antibody comprising thatsequence retains the ability to bind to fluorescein. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 108 or 116. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (i.e., in the FRs). Optionally, the anti-fluoresceinantibody comprises the VH sequence in SEQ ID NO: 108 or 116, includingpost-translational modifications of that sequence.

Another aspect as reported herein is an anti-theophylline antibody thatcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 68 or 76 or 84 or 92.In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-theophylline antibody comprisingthat sequence retains the ability to bind to theophylline. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 68 or 76 or 84 or 92. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (i.e., in the FRs). Optionally, the anti-theophyllineantibody comprises the VH sequence in SEQ ID NO: 68 or 76 or 84 or 92including post-translational modifications of that sequence.

Another aspect as reported herein is an anti-digoxigenin antibody,wherein the antibody comprises a VH as in any of the aspects and/orembodiments provided above, and a VL as in any of the aspects and/orembodiments provided above. In one embodiment, the antibody comprisesthe VH and VL sequences in SEQ ID NO: 04 or 12 or 20 or 28, and SEQ IDNO: 08 or 16 or 24 or 32, respectively, including post-translationalmodifications of those sequences.

Another aspect as reported herein is an anti-biotin antibody, whereinthe antibody comprises a VH as in any of the aspects and/or embodimentsprovided above, and a VL as in any of the aspects and/or embodimentsprovided above. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 36 or 44 or 52 or 60, and SEQ ID NO: 40 or 48 or56 or 64, respectively, including post-translational modifications ofthose sequences.

Another aspect as reported herein is an anti-fluorescein antibody,wherein the antibody comprises a VH as in any of the aspects and/orembodiments provided above, and a VL as in any of the aspects and/orembodiments provided above. In one embodiment, the antibody comprisesthe VH and VL sequences in SEQ ID NO: 108 or 116, and SEQ ID NO: 112 or120, respectively, including post-translational modifications of thosesequences.

Another aspect as reported herein is an anti-theophylline antibody,wherein the antibody comprises a VH as in any of the aspects and/orembodiments provided above, and a VL as in any of the aspects and/orembodiments provided above. In one embodiment, the antibody comprisesthe VH and VL sequences in SEQ ID NO: 68 or 76 or 84 or 92, and SEQ IDNO: 72 or 80 or 88 or 96, respectively, including post-translationalmodifications of those sequences.

A further position that was identified as modification point is theposition VH28 according to the Kabat numbering.

Replacement of the amino acid at position VH28 according to Kabat withCys generated antibody derivatives with heavy chain variable regionsequences that are listed as SEQ ID NO: 124 and 132 for anti-digoxigeninantibody-VH28C, SEQ ID NO: 156 and 164 for anti-theophyllineantibody-VH28C, SEQ ID NO: 140 and 148 for anti-biotin antibody-VH28C,and SEQ ID NO: 116 for anti-fluorescein antibody-VH28C.

ESI-MS analyses demonstrate that covalent antibody conjugation ofhaptenylated therapeutic peptides result in a conjugate of defined sizewhich is larger than non-complexed antibody or non-complexed peptide.

TABLE 1 TIC table. LC LC HC HC Conjugate sample Notes MW_(calc) MW_(exp)MW_(calc) MW_(exp) MW_(calc) Conjugate MW_(exp) humanized 1); 2337123371 49635 49634 n.a. n.a. anti- 2); 3) digoxigenin antibody humanized1); 23371 23371 49681 49680 n.a. n.a. anti- 2); 3) digoxigenin antibody-VH52bC Ac- 1); 23371 23371 49681 49680 152227 152233 PYY[PEG3- 2); 3)Cys(SS-R)- 4Abu-Dig] R = humanized anti- digoxigenin antibody VH52bCchimeric 2); 3) 23429 23429 49312 49311 n.a. n.a. anti-biotin antibodychimeric 2); 3) 23429 23429 49344 49343 n.a. n.a. anti-biotin antibodyVH53C humanized 1); 23465 23464 49218 49217 n.a. n.a. anti-biotin 2); 3)antibody humanized 1); 23465 23465 49250 49250 n.a. n.a. anti-biotin 2);3) antibody VH53C Ac- 2); 23429 23429 49344 49344 151233 151238PYY[PEG3- 3) Cys(SS-R)- βAla-Biot R = chimeric anti-biotin antibodyVH53C Ac- 2); 23429 23429 49344 49344 151381 151385 PYY[PEG3- 3)Cys(SS-R)- PEG2-Biot R = chimeric anti-biotin antibody VH53C Ac- 1);23465 23465 49250 49250 151118 151124 PYY[PEG3- 2); 3) Cys(SS-R)-βAla-Biot R = humanized anti-biotin antibody VH53C Ac- 1); 23465 2346549250 49250 151266 151272 PYY[PEG3- 2); 3) Cys(SS-R)- PEG2-Biot R =humanized anti-biotin antibody VH53C anti- 2); 23958 23958 49150 49149n.a. n.a. fluorescein 3) antibody anti- 2); 23958 23957 49124 49124 n.a.n.a. fluorescein 3) antibody VH52bC anti- 2); 23958 23957 49152 49151n.a. n.a. fluorescein 3) antibody VH28C Ac- 2); 23958 23957 49124 49125152271 152265 PYY[PEG3- 3) Cys(SS-R)- PEG2-Fluo R = anti- fluoresceinantibody VH52bC Ac- 2); 23958 23958 49152 49152 152324 152319 PYY[PEG3-3) Cys(SS-R)- PEG2-Fluo R = anti- fluorescein antibody VH28C 1) HC wN-terminal pyro-glutamic acid 2) HC w/o C-terminal Lys 3) HC w N -> D atGlycosylation site due to deglycosylation 4) LC w N-terminalpyro-glutamic acid

Antibody Affinity

In certain embodiments, the antibody as reported herein itself or theantibody in the complex as reported herein has a dissociation constant(Kd) of ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. of about10⁻⁸ M or less, e.g. from about 10⁻⁸M to about 10⁻¹³M, e.g., from about10⁻⁹M to about 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of FABs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol.Biol. 293 (1999) 865-881). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta, L. G. et al., Cancer Res. 57 (1997) 4593-4599). TheFab of interest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (about0.2 μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881. If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophotometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

Antibody Fragments

In certain embodiments, an antibody provided herein or in a conjugate asreported herein is an antibody fragment. Antibody fragments include, butare not limited to, Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments,and other fragments described below. For a review of certain antibodyfragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134. For areview of scFv fragments, see, e.g., Plueckthun, A., In; ThePharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore(eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion ofFab and F(ab′)₂ fragments comprising salvage receptor binding epitoperesidues and having increased in vivo half-life, see U.S. Pat. No.5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134; andHolliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J. et al.,Nat. Med. 9 (20039 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein or the antibody in aconjugate as reported herein is a chimeric antibody. Certain chimericantibodies are described, e.g., in U.S. Pat. No. 4,816,567; andMorrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855).In one example, a chimeric antibody comprises a non-human variableregion (e.g., a variable region derived from a mouse, rat, hamster,rabbit, or non-human primate, such as a monkey) and a human constantregion. In a further example, a chimeric antibody is a “class switched”antibody in which the class or subclass has been changed from that ofthe parent antibody. Chimeric antibodies include antigen-bindingfragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingSDR (a-CDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498(describing “resurfacing”); Dall'Acqua, W. F. et al., Methods 36 (2005)43-60 (describing “FR shuffling”); and Osbourn, J. et al., Methods 36(2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

Human Antibodies

In certain embodiments, an antibody provided herein or the antibody in aconjugate as reported herein is a human antibody. Human antibodies canbe produced using various techniques known in the art. Human antibodiesare described generally in van Dijk, M. A. and van de Winkel, J. G.,Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin.Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and US 2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147(1991) 86-95) Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Library-Derived Antibodies

Antibodies of the invention or antibodies in the conjugate as reportedherein may be isolated by screening combinatorial libraries forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37and further described, e.g., in the McCafferty, J. et al., Nature 348(1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks,J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. andBradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol.Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284(2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G. et al., Ann Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self-antigens without any immunization as described by Griffiths,A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and USPatent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

Antibody Formats

The above outlined antibodies and antibody fragments can be combined inmultiple ways to generate different antibody formats.

For example, one or more scFv antibody fragments can be fused to theC-terminus of one or more polypeptide chains of a complete antibody.Especially to each heavy chain C-terminus or to each light chainC-terminus a scFv antibody fragment can be fused.

For example, one or more antibody Fab fragments can be fused to theC-terminus of one or more polypeptide chains of a complete antibody.Especially to each heavy chain C-terminus or to each light chainC-terminus an antibody Fab fragment can be fused.

For example, one scFv and one antibody Fab fragment can be fused to theN-termini of an antibody Fc-region.

For example one scFv or antibody Fab fragment can be fused to anN-terminus of an antibody Fc-region and one scFv or antibody Fabfragment can be fused to the C-terminus of the respective other chain ofan antibody Fc-region.

Multispecific Antibodies

A wide variety of recombinant antibody formats have been developed, e.g.tetravalent bispecific antibodies by fusion of, e.g., an IgG antibodyformat and single chain domains (see e.g. Coloma, M. J., et al., NatureBiotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., NatureBiotech 25 (2007) 1233-1234).

Also several other formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N.,Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal ofImmunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech.25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFvs (Fischer, N. and Léger, O., Pathobiology74 (2007) 3-14). It has to be kept in mind that one may want to retaineffector functions, such as e.g. complement-dependent cytotoxicity (CDC)or antibody dependent cellular cytotoxicity (ADCC), which are mediatedthrough the Fc receptor binding, by maintaining a high degree ofsimilarity to naturally occurring antibodies.

In WO 2007/024715 are reported dual variable domain immunoglobulins asengineered multivalent and multispecific binding proteins. A process forthe preparation of biologically active antibody dimers is reported inU.S. Pat. No. 6,897,044. Multivalent FV antibody construct having atleast four variable domains which are linked with each over via peptidelinkers are reported in U.S. Pat. No. 7,129,330. Dimeric and multimericantigen binding structures are reported in US 2005/0079170. Tri- ortetra-valent monospecific antigen-binding protein comprising three orfour Fab fragments bound to each other covalently by a connectingstructure, which protein is not a natural immunoglobulin are reported inU.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecificantibodies are reported that can be efficiently expressed in prokaryoticand eukaryotic cells, and are useful in therapeutic and diagnosticmethods. A method of separating or preferentially synthesizing dimerswhich are linked via at least one interchain disulfide linkage fromdimers which are not linked via at least one interchain disulfidelinkage from a mixture comprising the two types of polypeptide dimers isreported in US 2005/0163782. Bispecific tetravalent receptors arereported in U.S. Pat. No. 5,959,083. Engineered antibodies with three ormore functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reportedin WO 1997/001580. WO 1992/004053 reports homoconjugates, typicallyprepared from monoclonal antibodies of the IgG class which bind to thesame antigenic determinant are covalently linked by syntheticcross-linking. Oligomeric monoclonal antibodies with high avidity forantigen are reported in

WO 1991/06305 whereby the oligomers, typically of the IgG class, aresecreted having two or more immunoglobulin monomers associated togetherto form tetravalent or hexavalent IgG molecules. Sheep-derivedantibodies and engineered antibody constructs are reported in U.S. Pat.No. 6,350,860, which can be used to treat diseases wherein interferongamma activity is pathogenic. In US 2005/0100543 are reported targetableconstructs that are multivalent carriers of bi-specific antibodies,i.e., each molecule of a targetable construct can serve as a carrier oftwo or more bi-specific antibodies. Genetically engineered bispecifictetravalent antibodies are reported in WO 1995/009917. In WO 2007/109254stabilized binding molecules that consist of or comprise a stabilizedscFv are reported.

In certain embodiments, an antibody provided herein or the antibody in aconjugate as reported herein is a multispecific antibody, e.g. abispecific antibody. Multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different sites. Incertain embodiments, one of the binding specificities is for a haptenand the other is for any other (non-hapten) antigen. Bispecificantibodies may also be used to localize cytotoxic agents to cells.Bispecific antibodies can be prepared as full length antibodies orantibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

In one embodiment the CH3 domains of the heavy chains of the bispecificantibody are altered by the “knob-into-holes” technology which isdescribed in detail with several examples in e.g. WO 96/027011, WO98/050431, Ridgway J. B., et al., Protein Eng. 9 (1996) 617-621,Merchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681. In thismethod the interaction surfaces of the two CH3 domains are altered toincrease the heterodimerization of both heavy chains containing thesetwo CH3 domains. Each of the two CH3 domains (of the two heavy chains)can be the “knob”, while the other is the “hole”. The introduction of adisulfide bridge stabilizes the heterodimers (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681, Atwell, S., et al. J. Mol. Biol. 270(1997) 26-35) and increases the yield.

In one embodiment of all aspects the bispecific antibody ischaracterized in that

-   -   the CH3 domain of one heavy chain and the CH3 domain of the        other heavy chain each meet at an interface which comprises an        original interface between the antibody CH3 domains,    -   wherein said interface is altered to promote the formation of        the bispecific antibody, wherein the alteration is characterized        in that        -   a) the CH3 domain of one heavy chain is altered,        -   so that within the original interface the CH3 domain of one            heavy chain that meets the original interface of the CH3            domain of the other heavy chain within the bispecific            antibody,        -   an amino acid residue is replaced with an amino acid residue            having a larger side chain volume, thereby generating a            protuberance within the interface of the CH3 domain of one            heavy chain which is positionable in a cavity within the            interface of the CH3 domain of the other heavy chain        -   and        -   b) the CH3 domain of the other heavy chain is altered,        -   so that within the original interface of the second CH3            domain that meets the original interface of the first CH3            domain within the bispecific antibody        -   an amino acid residue is replaced with an amino acid residue            having a smaller side chain volume, thereby generating a            cavity within the interface of the second CH3 domain within            which a protuberance within the interface of the first CH3            domain is positionable.

Thus, the antibodies as reported herein are in one embodimentcharacterized in that

-   -   the CH3 domain of the first heavy chain of the full length        antibody and the CH3 domain of the second heavy chain of the        full length antibody each meet at an interface which comprises        an alteration in the original interface between the antibody CH3        domains,    -   wherein i) in the CH3 domain of the first heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a larger side chain volume, thereby generating a        protuberance within the interface of the CH3 domain of one heavy        chain which is positionable in a cavity within the interface of        the CH3 domain of the other heavy chain    -   and wherein ii) in the CH3 domain of the second heavy chain    -   an amino acid residue is replaced with an amino acid residue        having a smaller side chain volume, thereby generating a cavity        within the interface of the second CH3 domain within which a        protuberance within the interface of the first CH3 domain is        positionable.

In one embodiment the amino acid residue having a larger side chainvolume is selected from the group consisting of arginine (R),phenylalanine (F), tyrosine (Y), tryptophan (W).

In one embodiment the amino acid residue having a smaller side chainvolume is selected from the group consisting of alanine (A), serine (S),threonine (T), valine (V).

In one embodiment both CH3 domains are further altered by theintroduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In one embodiment the bispecific antibody comprises a T366W mutation inthe CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations inthe CH3 domain of the “hole chain”. An additional interchain disulfidebridge between the CH3 domains can also be used (Merchant, A. M, et al.,Nature Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutationinto the CH3 domain of the “knobs chain” and a E356C mutation or a S354Cmutation into the CH3 domain of the “hole chain” (numbering according tothe EU index of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

In one embodiment the bispecific antibody comprises Y349C, T366Wmutations in one of the two CH3 domains and E356C, T366S, L368A, Y407Vmutations in the other of the two CH3 domains. In one embodiment thebispecific antibody comprises Y349C, T366W mutations in one of the twoCH3 domains and S354C, T366S, L368A, Y407V mutations in the other of thetwo CH3 domains (the additional Y349C mutation in one CH3 domain and theadditional E356C or S354C mutation in the other CH3 domain forming ainterchain disulfide bridge) (numbering according to EU index of Kabat;(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991))). Further knobs-in-holes technologies as described by EP 1870 459 A1, can be used alternatively or additionally. Thus anotherexample for the bispecific antibody are R409D, K370E mutations in theCH3 domain of the “knobs chain” and D399K, E357K mutations in the CH3domain of the “hole chain” (numbering according to EU index of Kabat;(Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991)).

In one embodiment the bispecific antibody comprises a T366W mutation inthe CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations inthe CH3 domain of the “hole chain” and additionally R409D, K370Emutations in the CH3 domain of the “knobs chain” and D399K, E357Kmutations in the CH3 domain of the “hole chain”.

In one embodiment the bispecific antibody comprises Y349C, T366Wmutations in one of the two CH3 domains and S354C, T366S, L368A, Y407Vmutations in the other of the two CH3 domains or the bispecific antibodycomprises Y349C, T366W mutations in one of the two CH3 domains andS354C, T366S, L368A, Y407V mutations in the other of the two CH3 domainsand additionally R409D, K370E mutations in the CH3 domain of the “knobschain” and D399K, E357K mutations in the CH3 domain of the “hole chain”.Such knob and hole mutations in the CH3 domain are typically used inhuman heavy chain constant regions of SEQ ID NO: 169, SEQ ID NO: 170,SEQ ID NO: 171, or SEQ ID NO: 172 (human IgG1 subclass allotypes(Caucasian and Afro-American or mutants L234A/L235A, andL234A/L235A/P329G), SEQ ID NO: 173, SEQ ID NO: 174, or SEQ ID NO: 175(human IgG4 subclass or mutants S228P, L235E, and S228P/L235E/P329G)(numbering according to the EU index of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)

In one embodiment the bispecific antibody comprises human heavy chainconstant regions of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, orSEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, or SEQ ID NO: 175further including such “knob” and “hole” mutations in the CH3 domain(e.g. Y349C, T366W mutations in one of the two CH3 domains and S354C,T366S, L368A, Y407V mutations in the other of the two CH3 domains)(numbering according to the EU index of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to a hapten as wellas another, different antigen (see US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

In one embodiment the first binding specificity of the bispecificantibody is to a hapten and the second binding specificity is to anon-hapten antigen. In one embodiment the non-hapten antigen is selectedfrom the leukocyte markers, CD2, CD3, CD4, CD5, CD6, CD7, CD8,CD11a,b,c, CD13, CD14, CD18, CD19, CD22, CD23, CD27 and its ligand, CD28and its ligands B7.1, B7.2, B7.3, CD29 and its ligand, CD30 and itsligand, CD40 and its ligand gp39, CD44, CD45 and isoforms, CD56, CD58,CD69, CD72, CTLA-4, LFA-1 and TCR; the histocompatibility antigens, MHCclass I or II, the Lewis Y antigens, SLex, SLey, SLea, and SLeb; theintegrins, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, αVβ3, and LFA-1,Mac-1, and p150,95, αVβ1, gpIIbIIIa, αR β3, α6β4, αVβ 5, αVβ6, and αV 627; the selectins, L-selectin, P-selectin, and E-selectin and theircounter receptors VCAM-1, ICAM-1, ICAM-2, and LFA-3; the interleukins,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, and IL-15; the interleukin receptor is selectedfrom the group consisting of IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R,IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, and IL-15R;the chemokine is selected from the group consisting of PF4, RANTES,MIPla, MCP1, NAP-2, Groa, GroP, and IL-8; the growth factor is selectedfrom the group consisting of TNFalpha, TGFbeta, TSH, VEGF/VPF, VEGFA,VEGFB, VEGF111, VEGF121, VEGF165, VEGF189, VEGF206, PTHrP, EGF family,PDGF family, endothelin, Fibrosin (FSF-1), human Laminin, and gastrinreleasing peptide (GRP), PLGF, HGH, HGHR; the growth factor receptor isselected from the group consisting of TNFalphaR, RGFbetaR, TSHR,VEGFR/VPFR, EGFR, PTHrPR, PDGFR family, EPO-R, GCSF-R and otherhematopoietic receptors; the interferon receptor is selected from thegroup consisting of IFNCαR, IFNβR, and IFNλR; the Ig and its receptor isselected from the group consisting of IgE, FcγRI, and FcγRII; the tumorantigen is selected from the group consisting of her2-neu, mucin, CEAand endosialin; the allergen is selected from the group consisting ofhouse dust mite antigen, lol p1 (grass) antigens, and urushiol; theviral polypeptide is selected from the group consisting of CMVglycoproteins B, H, and gCIII, HIV-1 envelope glycoproteins, RSVenvelope glycoproteins, HSV envelope glycoproteins, HPV envelopeglycoproteins, Hepatitis family surface antigens; the toxin is selectedfrom the group consisting of pseudomonas endotoxin andosteopontin/uropontin, snake venom, spider venom, and bee venomconotoxin; the blood factor is selected from the group consisting ofcomplement C3b, complement C4a, complement C4b-9, Rh factor, fibrinogen,fibrin, and myelin associated growth inhibitor; and the enzyme isselected from the group consisting of cholesterol ester transferpolypeptide, membrane bound matrix metalloproteases, and glutamic aciddecarboxylase (GAD).

Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), with theresulting variant VH or VL being tested for binding affinity. Affinitymaturation by constructing and reselecting from secondary libraries hasbeen described, e.g., in Hoogenboom, H. R. et al. in Methods inMolecular Biology 178 (2002) 1-37. In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. Heavy chain CDR3 and lightchain CDR3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein or comprised in aconjugate as reported herein is altered to increase or decrease theextent to which the antibody is glycosylated. Addition or deletion ofglycosylation sites to an antibody may be conveniently accomplished byaltering the amino acid sequence such that one or more glycosylationsites is created or removed.

Where the antibody comprises an Fc-region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of theFc-region. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32. The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to anFc-region. For example, the amount of fucose in such antibody may befrom 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc-region (EUnumbering of Fc-region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y.et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc-regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc-region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc-Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc-region of an antibody provided herein, therebygenerating an Fc-region variant. The Fc-region variant may comprise ahuman Fc-region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4Fc-region) comprising an amino acid modification (e.g. a substitution)at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc (RIII only, whereas monocytes express FcγRI, FcγRIIand FcγRIII. FcR expression on hematopoietic cells is summarized inTable 3 on page 464 of Ravetch, J. V. and Kinet, J. P., Annu. Rev.Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays toassess ADCC activity of a molecule of interest is described in U.S. Pat.No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci.USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad.Sci. USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann,M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, Wis.). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA95 (1998) 652-656. C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity.See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro, H. et al., J. Immunol.Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101 (2003)1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103 (2004)2738-2743). FcRn binding and in vivo clearance/half-life determinationscan also be performed using methods known in the art (see, e.g.,Petkova, S. B. et al., Int. Immunol. 18 (2006: 1759-1769).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc-region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, an antibody variant comprises an Fc-region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc-region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc-region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc-regionwith one or more substitutions therein which improve binding of theFc-region to FcRn. Such Fc variants include those with substitutions atone or more of Fc-region residues: 238, 256, 265, 272, 286, 303, 305,307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or434, e.g., substitution of Fc-region residue 434 (U.S. Pat. No.7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc-region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc-region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers,polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

Haptenylated Compounds

The hapten in a conjugate as reported herein may be conjugated, if it isnot by itself one of the molecules, to a therapeutic agent (drug), acytotoxic agent (e.g. a toxin such as doxorubicin or pertussis toxin), afluorophores such as a fluorescent dye like fluorescein or rhodamine, achelating agent for an imaging or radiotherapeutic metal, a peptidyl ornon-peptidyl label or detection tag, or a clearance-modifying agent suchas various isomers of polyethylene glycol, a peptide that binds to athird component, or another carbohydrate or lipophilic agent. Such aconjugate is denoted as haptenylated compound. The conjugation can beeither directly or via an intervening linker.

a) Therapeutic Moieties

The drug moiety (D) of the hapten-drug conjugate (ADC, haptenylateddrug) can be any compound, moiety or group which has a cytotoxic orcytostatic effect. Drug moieties include: (i) chemotherapeutic agents,which may function as microtubule inhibitors, mitosis inhibitors,topoisomerase inhibitors, or DNA intercalators; (ii) protein toxins,which may function enzymatically; and (iii) radioisotopes.

Exemplary drug moieties include, but are not limited to, a maytansinoid,an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicinand other enediyne antibiotics, a taxane, an anthracycline, andstereoisomers, isosters, analogs or derivatives thereof.

Protein toxins include diphtheria-A chain, non-binding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain (Vitetta et al (1987) Science, 238:1098), abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-5),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes (WO 93/21232).

Therapeutic radioisotopes include 32P, 33P, 90Y, 125I, 131I, 131In,153Sm, 186Re, 188Re, 211At, 212B, 212Pb, and radioactive isotopes of Lu.

The radioisotope or other labels may be incorporated in known ways(Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57;“Monoclonal Antibodies in Immunoscintigraphy” Chatal, CRC Press 1989).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of a radionuclide to the complex (WO 94/11026).

b) Labels

The haptenylated compound can be a haptenylated label. Any label moietywhich can be covalently attached to the hapten can be used (see e.g.Singh et al (2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D.(1999) Using Antibodies: A Laboratory Manual, Cold Springs HarborLaboratory Press, Cold Spring Harbor, N.Y.; Lundblad R. L. (1991)Chemical Reagents for Protein Modification, 2nd ed. CRC Press, BocaRaton, Fla.). The label may function to: (i) provide a detectablesignal; (ii) interact with a second label to modify the detectablesignal provided by the first or second label, e.g. to give FRET(fluorescence resonance energy transfer); (iii) affect mobility, e.g.electrophoretic mobility or cell-permeability, by charge,hydrophobicity, shape, or other physical parameters, or (iv) provide acapture moiety, e.g. to modulate ionic complexation.

Conjugates comprising a haptenylated label as reported herein may beuseful in diagnostic assays, e.g., for detecting expression of anantigen of interest in specific cells, tissues, or serum. For diagnosticapplications, a bispecific antibody will be used wherein the firstbinding specificity binds to a target and the second binding specificitybinds to a haptenylated label. The hapten will typically be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S,64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe,177Lu, 211At, or 131Bi. Radioisotope labeled conjugates are useful inreceptor targeted imaging experiments. The antigen (hapten) can belabeled with ligand reagents that bind, chelate or otherwise complex aradioisotope metal using the techniques described in Current Protocolsin Immunology, (1991) Volumes 1 and 2, Coligen et al, Ed.Wiley-Interscience, New York, N.Y., Pubs. Chelating ligands which maycomplex a metal ion include DOTA, DOTP, DOTMA, DTPA and TETA(Macrocyclics, Dallas, Tex.). Radionuclides can be targeted viacomplexation with the complex as reported herein (Wu et al, NatureBiotechnology 23(9) (2005) 1137-1146). Receptor target imaging withradionuclide labeled complexes can provide a marker of pathwayactivation by detection and quantitation of progressive accumulation ofcomplexes or corresponding therapeutic antibodies in tumor tissue(Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210).

Metal-chelate complexes suitable as labels for imaging experiments (US2010/0111856; U.S. Pat. Nos. 5,342,606; 5,428,155; 5,316,757; 5,480,990;5,462,725; 5,428,139; 5,385,893; 5,739,294; 5,750,660; 5,834,456;Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al,Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al, Bioconjugate Chem. 1(1990) 59-65; Meares et al, J. Cancer (1990), Suppl. 10:21-26; Izard etal, Bioconjugate Chem. 3 (1992) 346-350; Nikula et al, Nucl. Med. Biol.22 (1995) 387-90; Camera et al, Nucl. Med. Biol. 20 (1993) 955-62; Kukiset al, J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44(2003) 1663-1670; Camera et al, J. Nucl. Med. 21 (1994) 640-646; Ruegget al, Cancer Res. 50 (1990) 4221-4226; Verel et al, J. Nucl. Med. 44(2003) 1663-1670; Lee et al, Cancer Res. 61 (2001) 4474-4482; Mitchell,et al, J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al BioconjugateChem. 10 (1999) 103-111; Miederer et al, J. Nucl. Med. 45 (2004)129-137; DeNardo et al, Clinical Cancer Research 4 (1998) 2483-90; Blendet al, Cancer Biotherapy & Radiopharmaceuticals 18 (2003) 355-363;Nikula et al J. Nucl. Med. 40 (1999) 166-76; Kobayashi et al, J. Nucl.Med. 39 (1998) 829-36; Mardirossian et al, Nucl. Med. Biol. 20 (1993)65-74; Roselli et al, Cancer Biotherapy & Radiopharmaceuticals, 14(1999) 209-20).

(b) Fluorescent labels such as rare earth chelates (europium chelates),fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansyl; Lissamine;cyanines; phycoerythrins; Texas Red; and analogs thereof. Thefluorescent labels can be conjugated to the antigen (hapten) using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescent dyes and fluorescent label reagents include thosewhich are commercially available from Invitrogen/Molecular Probes(Eugene, Oreg., USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

Detection labels such as fluorescent dyes and chemiluminescent dyes(Briggs et al “Synthesis of Functionalised Fluorescent Dyes and TheirCoupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1(1997) 1051-1058) provide a detectable signal and are generallyapplicable for labeling, especially with the following properties: (i)the labeled conjugate should produce a very high signal with lowbackground so that small quantities of conjugate can be sensitivelydetected in both cell-free and cell-based assays; and (ii) the labeledconjugate should be photostable so that the fluorescent signal may beobserved, monitored and recorded without significant photo bleaching.For applications involving cell surface binding of labeled conjugates tomembranes or cell surfaces, especially live cells, the labels should(iii) have good water-solubility to achieve effective conjugateconcentration and detection sensitivity and (iv) are non-toxic to livingcells so as not to disrupt the normal metabolic processes of the cellsor cause premature cell death.

(c) Various enzyme-substrate labels are available or disclosed (see e.g.U.S. Pat. No. 4,275,149). The enzyme generally catalyzes a chemicalalteration of a chromogenic substrate that can be measured using varioustechniques. For example, the enzyme may catalyze a color change in asubstrate, which can be measured spectrophotometrically. Alternatively,the enzyme may alter the fluorescence or chemiluminescence of thesubstrate. The chemiluminescent substrate becomes electronically excitedby a chemical reaction and may then emit light which can be measured(using a chemiluminometer, for example) or donates energy to afluorescent acceptor. Examples of enzymatic labels include luciferases(e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP),alkaline phosphatase (AP), (3-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to polypeptides are describedin O'Sullivan et al “Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed. byJ. Langone & IT Van Vunakis), Academic Press, New York, 73 (1981)147-166.

Examples of enzyme-substrate combinations (U.S. Pat. Nos. 4,275,149;4,318,980) include, for example:

(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) (3-D-galactosidase ((3-D-Gal) with a chromogenic substrate (e.g.,p-nitro phenyl-(3-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-(3-D-galactosidase.

The labeled conjugate as reported herein may be employed in any knownassay method, such as ELISA, competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Zola,Monoclonal Antibodies: A Manual of Techniques (1987) pp. 147-158, CRCPress, Inc.).

Labeled conjugates as reported herein are useful as imaging biomarkersand probes by the various methods and techniques of biomedical andmolecular imaging such as: (i) MRI (magnetic resonance imaging); (ii)MicroCT (computerized tomography); (iii) SPECT (single photon emissioncomputed tomography); (iv) PET (positron emission tomography) Tinianow,J. et al Nuclear Medicine and Biology, 37(3) (2010) 289-297; Chen et al,Bioconjugate Chem. 15 (2004) 41-49; US 2010/0111856 (v) bioluminescence;(vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is animaging procedure in which conjugates labeled with radioactivesubstances are administered to an animal or human patient and a pictureis taken of sites in the body where the conjugate localizes (U.S. Pat.No. 6,528,624). Imaging biomarkers may be objectively measured andevaluated as an indicator of normal biological processes, pathogenicprocesses, or pharmacological responses to a therapeutic intervention.Biomarkers may be of several types: Type 0 markers are natural historymarkers of a disease and correlate longitudinally with known clinicalindices, e.g. MRI assessment of synovial inflammation in rheumatoidarthritis; Type I markers capture the effect of an intervention inaccordance with a mechanism-of-action, even though the mechanism may notbe associated with clinical outcome; Type II markers function assurrogate endpoints where the change in, or signal from, the biomarkerpredicts a clinical benefit to “validate” the targeted response, such asmeasured bone erosion in rheumatoid arthritis by CT. Imaging biomarkersthus can provide pharmacodynamic (PD) therapeutic information about: (i)expression of a target protein, (ii) binding of a therapeutic to thetarget protein, i.e. selectivity, and (iii) clearance and half-lifepharmacokinetic data. Advantages of in vivo imaging biomarkers relativeto lab-based biomarkers include: non-invasive treatment, quantifiable,whole body assessment, repetitive dosing and assessment, i.e. multipletime points, and potentially transferable effects from preclinical(small animal) to clinical (human) results. For some applications,bioimaging supplants or minimizes the number of animal experiments inpreclinical studies.

Peptide labeling methods are well known. See Haugland (2003) MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc.; Brinkley (1992) Bioconjugate Chem. 3:2; Garman, (1997)Non-Radioactive Labeling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Glazer et al Chemical Modificationof Proteins. Laboratory Techniques in Biochemistry and Molecular Biology(T. S. Work and E. Work, Eds.) American Elsevier Publishing Co., NewYork; Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents forProtein Modification, Vols. I and II, CRC Press, New York; Pfleiderer,G. (1985) “Chemical Modification of Proteins”, Modern Methods in ProteinChemistry, H. Tschesche, Ed., Walter DeGruyter, Berlin and New York; andWong (1991) Chemistry of Protein Conjugation and Cross-linking, CRCPress, Boca Raton, Fla.); DeLeon-Rodriguez et al, Chem. Eur. J. 10(2004) 1149-1155; Lewis et al, Bioconjugate Chem. 12 (2001) 320-324; Liet al, Bioconjugate Chem. 13 (2002) 110-115; Mier et al BioconjugateChem. 16 (2005) 240-237.

Antibody Conjugates

The antibody in a conjugate as reported herein may be furtherconjugated, if it is not by itself one of the molecules, to atherapeutic agent (drug), a cytotoxic agent (e.g. a toxin such asdoxorubicin or pertussis toxin), a fluorophores such as a fluorescentdye like fluorescein or rhodamine, a chelating agent for an imaging orradiotherapeutic metal, a peptidyl or non-peptidyl label or detectiontag, or a clearance-modifying agent such as various isomers ofpolyethylene glycol, a peptide that binds to a third component, oranother carbohydrate or lipophilic agent.

Immunoconjugates

The invention also provides immunoconjugates comprising an antibody asreported herein or a conjugate as reported herein conjugated to one ormore cytotoxic agents, such as chemotherapeutic agents or drugs, growthinhibitory agents, toxins (e.g., protein toxins, enzymatically activetoxins of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos.5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin orderivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116,5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman, L. M.et al., Cancer Res. 53 (1993) 3336-3342; and Lode, H. N. et al., CancerRes. 58 (1998) 2925-2928); an anthracycline such as daunomycin ordoxorubicin (see Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523;Jeffrey, S. C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362;Torgov, M. Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. etal., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G. M. etal., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H. D. etal., J. Med. Chem. 45 (2002) 4336-4343; and U.S. Pat. No. 6,630,579);methotrexate; vindesine; a taxane such as docetaxel, paclitaxel,larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein or a complex as reported herein conjugated to aradioactive atom to form a radioconjugate. A variety of radioactiveisotopes are available for the production of radioconjugates. Examplesinclude At²¹¹, I³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, B²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the radioconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example TC^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, MRI),such as iodine-123 again, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and a cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas his (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta, E. S. et al., Science 238 (1987)1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari, R. V. et al., Cancer Res. 52 (1992)127-131; U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

Linker

The term “linker” denotes a bifunctional or multifunctional moiety whichcan be used to conjugate (link) the antigen (e.g. a hapten) to othermoieties, such as detectable labels or drugs. Antigen (hapten)conjugates can be conveniently prepared using a linker having reactivefunctionality for binding to the drug, to the antigen (hapten) and tothe anti-hapten antibody.

In one embodiment, a linker has a reactive site which has anelectrophilic group that is reactive to a nucleophilic group present onthe anti-hapten antibody. A cysteine thiol group on the antibody forexample is reactive with an electrophilic group on a linker and forms acovalent bond to a linker. Useful electrophilic groups include, but arenot limited to, another thiol, maleimide and haloacetamide groups (seee.g. conjugation method at page 766 of Klussman et al, BioconjugateChemistry 15(4) (2004) 765-773).

Examples of thiol-reaction functional groups include, but are notlimited to, thiol, maleimide, alpha-haloacetyl, activated esters such assuccinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates.

The linker may comprise amino acid residues which link the antigen(hapten) to the payload. The amino acid residues may form a dipeptide,tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide,octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptideunit. Amino acid residues include those occurring naturally, as well asnon-naturally occurring amino acid analogs, such as e.g. citrulline orβ-amino acids, such as e.g. β-alanine, or ω-amino acids such as4-amino-butyric acid.

In another embodiment, the linker has a reactive functional group whichhas a nucleophilic group that is reactive to an electrophilic grouppresent on the antigen (hapten) or the antibody (anti-hapten antibody).Useful electrophilic groups include, but are not limited to, aldehydeand ketone carbonyl groups. The heteroatom of a nucleophilic group of alinker can react with an electrophilic group on the hapten or theantibody and form a covalent bond to an antigen (hapten) or theantibody. Useful nucleophilic groups on a linker include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on anantigen (hapten) provides a convenient site for attachment to a linker.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schroder and K. Lubke “The Peptides”, volume1 (1965) 76-136, Academic Press) which is well known in the field ofpeptide chemistry.

In another embodiment, the linker may be substituted with groups whichmodulated solubility or reactivity. For example, a charged substituentsuch as sulfonate (SO₃ ⁻) or ammonium or a polymer such as PEG, mayincrease water solubility of the reagent and facilitate the couplingreaction of the linker reagent with the antigen (hapten) or the drugmoiety, or facilitate the coupling reaction depending on the syntheticroute employed.

The conjugates comprising a drug or label as reported herein expresslycontemplate, but are not limited to, complexes prepared with linkerreagents: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄, which arecommercially available from Pierce Biotechnology, Inc. Bis-maleimidereagents allow the attachment of e.g. a thiol group to athiol-containing drug moiety, label, or linker intermediate, in asequential or concurrent fashion. Other functional groups besidesmaleimide, which are reactive with e.g. a thiol group includeiodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Exemplary linker include a valine-citrulline (val-cit or vc) dipeptidelinker reagent having a maleimide stretcher and apara-aminobenzylcarbamoyl (PAB) self-immolative spacer, and aphe-lys(Mtr) dipeptide linker reagent having a maleimide Stretcher unitand a p-amino benzyl self-immolative spacer.

Cysteine thiol groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker reagents andhaptenylated compounds including: (i) active esters such as NHS esters,HOBt esters, haloformates, and acid halides; (ii) alkyl and benzylhalides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups; and (iv) disulfides, including pyridyl disulfides, viasulfide exchange. Nucleophilic groups on a haptenylated compoundinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

III. Nucleic Acid

The DNA encoding the amino acid sequence variant of the antibody asreported herein or as comprised in a conjugate as reported herein can beprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may be constructed also byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. Mutagenic primers encode the cysteine codonreplacement(s). Standard mutagenesis techniques can be employed togenerate DNA encoding such modified engineered antibodies. Generalguidance can be found in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel et al Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

IV. Expression and Purification

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an antibody described herein is provided.Such nucleic acid may encode an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,Sp20 cell). In one embodiment, a method of making an antibody asreported herein is provided, wherein the method comprises culturing ahost cell comprising a nucleic acid encoding the antibody, as providedabove, under conditions suitable for expression of the antibody, andoptionally recovering the antibody from the host cell (or host cellculture medium).

For recombinant production of an antibody as reported herein, nucleicacid encoding an antibody, e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gemgross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li,H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

V. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the antibodies, especially the bispecificantibodies, and conjugates as reported herein is useful for detectingthe presence of one or more target molecules in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In one embodiment a biological sample comprises a cell ortissue.

In one embodiment, an antibody or conjugate as reported herein for usein a method of diagnosis or detection is provided. In certainembodiments, the method comprises contacting the biological sample withan antibody or conjugate as reported herein under conditions permissivefor binding of the antibody or the conjugate to the target, anddetecting whether a complex is formed between the antibody or theconjugate and the target. Such method may be an in vitro or in vivomethod.

In certain embodiments, labeled antibodies or conjugates are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

VI. Pharmaceutical Formulations

Pharmaceutical formulations of an antibody or conjugate as reportedherein are prepared by mixing such antibody or conjugate having thedesired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences, 16th edition,Osol, A. (ed.) (1980)), in the form of lyophilized formulations oraqueous solutions. Pharmaceutically acceptable carriers are generallynontoxic to recipients at the dosages and concentrations employed, andinclude, but are not limited to: buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyl dimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone);amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody or conjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

VII. Therapeutic Methods and Compositions

Any of the antibodies or conjugates reported herein may be used intherapeutic methods.

In one aspect, an antibody or a conjugate as reported herein for use asa medicament is provided. In further aspects, an antibody or a conjugateas reported herein for use in treating a disease is provided. In certainembodiments, an antibody or a conjugate as reported herein for use in amethod of treatment is provided. In certain embodiments, the inventionprovides an antibody or a conjugate as reported herein for use in amethod of treating an individual comprising administering to theindividual an effective amount of the antibody or the conjugate asreported herein. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides for the use of an antibodyor a conjugate as reported herein in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment of adisease. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual having adisease an effective amount of the medicament. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such a disease an effective amount of an antibody or aconjugate as reported herein. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the antibodies or conjugates as reported herein, e.g.,for use in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the antibodies or conjugatesas reported herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the antibodiesor conjugates as reported herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies and conjugates as reported herein can be used either alone orin combination with other agents in a therapy. For instance, an antibodyor conjugate as reported herein may be co-administered with at least oneadditional therapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies and conjugates as reportedherein can also be used in combination with radiation therapy.

An antibody or conjugate as reported herein (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or conjugates as reported herein would be formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of the disorder,the site of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The antibody or conjugate need not be, but is optionallyformulated with one or more agents currently used to prevent or treatthe disorder in question. The effective amount of such other agentsdepends on the amount of antibody or conjugate present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or conjugate as reported herein (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orconjugate, the severity and course of the disease, whether the antibodyor conjugate is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody or conjugate, and the discretion of the attending physician.The antibody or conjugate is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody or conjugate can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody or conjugate would be inthe range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or moredoses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses may be administered. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an antibody or a conjugate as reportedherein.

VIII. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or a complex as reported herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises an antibody or a complex as reported herein; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an antibody or a conjugate as reported herein.

The disclosure of all references cited herein is herewith incorporatedby reference.

The following examples, figures and sequences are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

EXAMPLES Example 1 Isolation and Characterization of cDNAs Encoding theVH and VL Domains of a Murine Anti-Digoxigenin Antibody and a MurineAnti-Biotin Antibody of IgG1 Class with Kappa Light Chain from MouseHybridoma

The isolation and characterization of cDNAs encoding the VH and VLdomains of anti-digoxigenin antibodies, the RNA preparation, generationof DNA fragments, the cloning of the DNA fragments into plasmids and thedetermination of the DNA- and amino acid sequences were described in WO2011/003557 and WO 2011/003780, respectively.

The protein and (DNA) sequence information of the VH and VL domains ofthe murine hapten-binding antibodies were obtained directly fromhybridoma clones. The experimental steps performed subsequently were (i)the isolation of RNA from antibody producing hybridoma cells, (ii)conversion of this RNA into cDNA, the transfer into VH and VL harboringPCR fragments, and (iii) integration of these PCR fragments intoplasmids vectors for propagation in E. coli and determination of theirDNA (and deduced protein) sequences.

RNA Preparation from Hybridoma Cells:

RNA was prepared from 5×10⁶ antibody expressing hybridoma cells applyingthe RNeasy-Kit (Qiagen). Briefly, the sedimented cells were washed oncein PBS and sedimented and subsequently resuspended for lysis in 500 μlRLT-buffer (+β-ME). The cells were completely lysed by passing through aQiashredder (Qiagen) and then subjected to the matrix-mediatedpurification procedure (ETOH, RNeasy columns) as described in themanufacturer's manual. After the last washing step, RNA was recoveredfrom the columns in 50 μl RNAse-free water. The concentration of therecovered RNA was determined by quantifying A260 and A280 of 1:20diluted samples. The integrity (quality, degree of degradation) of theisolated RNA samples was analyzed by denaturing RNA gel electrophoresison Formamide-Agarose gels (see Maniatis Manual). Discrete bandsrepresenting the intact 18s and 28 s ribosomal RNAs were obtained andintactness (and approx. 2:1 intensity ratios) of these bands indicated agood quality of the RNA preparations. The isolated RNAs from hybridomawere frozen and stored at −80° C. in aliquots.

Generation of DNA Fragments Encoding VH and VH by RACE PCR, Cloning ofthese DNA Fragments into Plasmids and Determination of their DNA- andAmino Acid Sequences

The cDNA for subsequent (RACE-) PCR reactions were prepared from RNApreparations by applying the technologies as described in Internationalpatent application PCT/EP2011/074273. Subsequently, the VH andVL-encoding PCR fragments were isolated by agarose gel extraction andsubsequent purification by standard molecular biology techniques.PWO-generated purified PCR fragments were inserted into the vector pCRbluntII topo by applying the pCR bluntII topo Kit (Invitrogen) exactlyfollowing the manufacturer's instructions. The Topo-ligation reactionswere transformed into E. coli Topo10-one-shot competent cells.Thereafter, E. coli clones that contained vectors with either VL- or VHcontaining inserts were identified as colonies on LB-Kanamycin agarplates. Plasmids were prepared from these colonies and the presence ofthe desired insert in the vector was confirmed by restriction digestionwith EcoRI. Because the vector backbone contains EcoRI restrictionrecognition sites flanking each side of the insert, plasmids harboringinserts were defined by having EcoRI-releasable inserts of approx. 800bp (for VL) or 600 bp (for VH). The DNA sequence and the deduced proteinsequence of the VL and VH were determined by automated DNA sequencing onmultiple clones for VH and VL.

The murine VL sequence of the anti-biotin antibody is depicted in SEQ IDNO: 40. The murine VH sequence of the anti-biotin antibody is depictedin SEQ ID NO: 36. The murine VL sequence of the anti-digoxigeninantibody is depicted in SEQ ID NO: 08. The murine VH sequence of theanti-digoxigenin antibody is depicted in SEQ ID NO: 04.

Example 2 Isolation and Characterization of cDNAs Encoding the VH and VLDomains of a Murine Anti-Theophylline Antibody of IgG1 Class with KappaLight Chain from Mouse Hybridoma

The sequences of the anti-theophylline antibody were obtained asoutlined in Example 1.

The murine VL sequence of the anti-theophylline antibody is depicted inSEQ ID NO: 72. The murine VH sequence of the anti-theophylline antibodyis depicted in SEQ ID NO: 68.

Example 3 Humanization of the VH and VL Domains of MurineAnti-Digoxigenin Antibody and Anti-Biotin Antibody

The generation of humanized variants of the digoxigenin-binding antibodyhas been described in detail in WO 2011/003557 and WO 2011/003780. Themurine biotin-binding antibody muM33 was humanized in a similar manneras follows:

The generation and characterization of encoding sequences and amino acidsequences that comprise the VH and VL domains of a murine anti-biotinantibody of the IgG1 class with kappa light chain from mouse hybridomaare described in WO 2011/003557 and WO 2011/003780. Based on thisinformation, a corresponding humanized anti-biotin antibody wasgenerated (huM33) based on the human germline framework IGHV1-69-02 andIGKV1-27-01 combination. For VL, it was not necessary to integrate anybackmutation in the framework of the human IGKV1-27-01 and the human Jelement of the IGKJ2-01 germline. The humanized VH is based on the humanIGHV1-69-02 germline and the human J element of the IGHJ4-01-3 germlineTwo backmutations in framework region 1 at position 24 (A24S) and inframework region 3 at position 73 (K73T) were introduced. The amino acidsequence of the humanized VH is depicted in SEQ ID NO: 44 and the aminoacid sequence of the humanized VL is shown in SEQ ID NO: 48.

Example 4 Humanization of the VH and VL Domains of the MurineAnti-Theophylline Antibody

The murine theophylline-binding antibody was humanized as follows: ahumanized anti-theophylline antibody was generated based on the humangermline framework IGHV4-31-02 and IGKV2-30-01 combination. Thehumanized VH is based on the human IGHV4-31-02 germline and the human Jelement of the IGHJ4-01-3 germline One backmutations in framework region3 at position 71 (V71R) was introduced. The humanized VL is based on thehuman IGHV2-30-01 germline and the human J element of the IGKJ2-01germline. One backmutations in framework region 2 at position 46 (R46L)was introduced. The amino acid sequence of the humanized VH is depictedin SEQ ID NO: 76 and the amino acid sequence of the humanized VL isshown in SEQ ID NO: 80.

Example 5 Crystallization and X-Ray Structure Determination of theBinding Region of the Murine Anti-Digoxigenin Fv Region in the Presenceof Digoxigenin, and of the Binding Region of the Murine Anti-Biotin FvRegion in the Presence of Biotin

The determination of the structure of the Fab fragment of thedigoxigenin-binding antibody has been described in detail in WO2011/003557 and WO 2011/003780, also published (3RA7) in Metz, S. etal., Proc. Natl. Acad. Sci. USA 108 (2011) 8194-8199.

The structure of the murine anti-biotin antibody was determined.Therefore, Fab fragments were generated by protease digestion of thepurified IgGs and subsequently purified, applying well known state ofthe art methods (papain digestion).

For crystallization of the apo Fab fragment (purified Fabs) in 20 mMHis-HCl, 140 mM NaCl, pH 6.0 were concentrated to 13 mg/ml.Crystallization droplets were set up at 21° C. by mixing 0.2 μl ofprotein solution with 0.2 μl reservoir solution in vapor diffusionsitting drop experiments. Crystals appeared out of 0.1 M Tris pH 8.5,0.01 M cobalt chloride, 20% polyvinylpyrrolidone K15 within 5 days andgrew to a final size of 0.3 mm×0.06 mm×0.03 mm within 8 days.

Crystals were harvested with 15% Glycerol as cryoprotectant and thenflash frozen in liquid N2. Diffraction images were collected with aPilatus 6M detector at a temperature of 100K at the beam line X10SA ofthe Swiss Light Source and processed with the programs XDS (Kabsch, W.,J. Appl. Cryst. 26 (1993) 795-800) and scaled with SCALA (obtained fromBRUKER AXS), yielding data to 2.22 Å resolution. This Fab fragmentcrystal belongs to monoclinic space group P21 with cell dimensions ofa=90.23 Å b=118.45 Å c=96.79 Å and β=117.53° and contains four Fabmolecules per crystallographic asymmetric unit (see Table 3).

Standard crystallographic programs from the CCP4 software suite wereused to solve the structure by molecular replacement with the PDB entry3PQP as search model, to calculate the electron density, and to refinethe x-ray structure (CCP4, Collaborative Computational Project, ActaCrystallogr. D, 760-763 (1994)). The structural models were rebuilt intothe electron density using COOT (Emsley, P., et al. Acta Crystallogr. DBiol. Crystallogr. 60 (2010) 486-501). Coordinates were refined withREFMAC5 (Murshudov, G. N., et al. Acta Crystallogr. D Biol. Crystallogr.53 (1997) 240-55) and with autoBUSTER (Global Phasing Ltd.).

TABLE 3 Data collection and structure refinement statistics formonoclinic muM33 Fab fragment apo-crystal Data Collection Wavelength (Å)1.0 Resolution¹ (Å)     2.22 (2.34-2.22) Unique reflections¹ 77716(11301) Completeness (%)¹ 98.0 (100)  R_(merge) (%)^(1,2)  6.4 (44.4)<I/σ>¹ 8.3 (1.7) Unit Cell (Space group C2) a = 90.23 Å b = 118.45 Å c =96.73 Å and β = 117.53° Refinement Resolution (Å)     2.2 (2.28-2.22)R_(cryst) ^(1,3)  20.66 (21.84)) R_(free) ^(1,4) 25.23 (26.47) Number ofAtoms in 13314 refinement R.m.s. deviations from ideality 0.01/1.21 Bondlengths (Å)/angles (°) Main chain dihedral angles (%) 90.4/9.1/0.3/0.2Most favored/allowed/generous/ disallowed⁵ ¹Values in parentheses referto the highest resolution bins. ²R_(merge) = Σ|I − <I>|/ΣI where I isintensity. ³R_(cryst) = Σ|F_(o) − <F_(c)>|/ΣF_(o) where F_(o) is theobserved and F_(c) is the calculated structure factor amplitude.⁴R_(free) was calculated based on 5% of the total data omitted duringrefinement. ⁵Calculated with PROCHECK [Laskowski, R. A., et al., J.Appl. Crystallogr. 26, 283-291 (1993)].

For the crystallization of Fab-fragment in complex with abiotin-derivative, apo Crystals of the Fab fragment used for soakingexperiments were derived out of 0.8 M Succinic Acid within 3 days afterscreening and grew to a final size of 0.25 mm×0.04 mm×0.04 mm within 5days. Biocytinamid was dissolved at 100 mM in water. Subsequently, thecompound was diluted to 10 mM working concentration in crystallizationsolution and applied to the crystals in the crystallization droplet.Crystals were washed three times with 2 μl of 10 mM compound solutionand were finally incubated for 16 h with biocytinamid at 21° C.

Crystals were harvested with 15% glycerol as cryoprotectant and thenflash frozen in liquid N₂. Diffraction images were collected with aPilatus 6M detector at a temperature of 100 K at the beam line X10SA ofthe Swiss Light Source and processed with the programs XDS (Kabsch, W.,J. Appl. Cryst. 26 (1993) 795-800) and scaled with SCALA (obtained fromBRUKER AXS), yielding data to 2.35 Å resolution. This Fab fragmentcrystal belongs to monoclinic space group P21 with cell dimensions ofa=89.09 Å b=119.62 Å c=96.18 Å and β=117.15° and contains four Fabmolecules per crystallographic asymmetric unit (see Table 4).

Standard crystallographic programs from the CCP4 software suite wereused to solve the structure by molecular replacement with thecoordinates of the apo Fab fragment as search model, to calculate theelectron density, and to refine the x-ray structure to a resolution of2.5 Å (CCP4). The structural models were rebuilt into the electrondensity using COOT Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K.Features and development of COOT. Acta Crystallogr. D Biol. Crystallogr.60, 486-501 (2010)). Coordinates were refined with REFMAC5 (Murshudov,G. N., et al. Acta Crystallogr. D Biol. Crystallogr. 53, 240-255 (1997))and with autoBUSTER (Global Phasing Ltd.).

TABLE 4 Data collection and structure refinement statistics formonoclinic muM33 Fab fragment biocytinamid complex crystal DataCollection Wavelength (Å) 1.0 Resolution¹ (Å)     2.35 (2.45-2.35)Unique reflections¹ 74645 (8714)  Completeness (%)¹ 99.9 (99.9)R_(merge) (%)^(1,2)  6.30 (65.00) <I/σ>¹ 10.29 (1.18)  Unit Cell (Spacegroup C2) a = 89.09 Å b = 119.62 Å c = 96.18 Å and β = 117.15°Refinement Resolution (Å)      2.5 (2.565-2.500) R_(cryst) ^(1,3)  20.92(36.86)) R_(free) ^(1,4) 27.56 (47.5)  Number of Atoms in refinement13656 R.m.s. deviations from ideality 0.009/1.43 Bond lengths (Å)/angles(°) Main chain dihedral angles (%) 87.5/12.0/0.2/0.3 Mostfavored/allowed/generous/ disallowed⁵ ¹Values in parentheses refer tothe highest resolution bins. ²R_(merge) = Σ|I − <I>|/ΣI where I isintensity. ³R_(cryst) = Σ|F_(o) − <F_(c)>|/ΣF_(o) where F_(o) is theobserved and F_(c) is the calculated structure factor amplitude.⁴R_(free) was calculated based on 5% of the total data omitted duringrefinement. ⁵Calculated with PROCHECK [Laskowski, R. A., Mac Arthur, M.W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check thestereochemical quality of protein structure. J. Appl. Crystallogr. 26,283-291 (1993)].

The result of the experimental structure determination is shown in FIG.33. The crystal form of the complex contained four independentbiocytinamid:anti-biotin Fab complexes in the asymmetric unit, withbiocytinamid bound similarly by all Fab molecules. Biocytidinamide isbound in a pocket formed by CDRs 1 and 3 of the heavy chain and all 3light chain CDRs. The binding pocket of the ligand is defined byresidues ASN29, ASP31, THR32, PHE33, GLN35, TRP99 and TRP106 from theheavy chain and ASN31, TYR32, LEU33, SER34, TYR49, SER50, PHE91 andTYR96 from the light chain. The biotin head group forms hydrogen bondswith residues of CDR2 and CDR1 at one end of the pocket: N3 ofbiocytinamid is interacting with the hydroxyl-oxygen of Ser50 whereasO22 is in contact with the backbone-amide nitrogen of the same residue.In addition, O22 of biocytinamid is also hydrogen-bonded to thehydroxyl-group oxygen of Ser34. In addition to that, hydrophobicinteractions are observed between biocytinamid and the aromatic sidechains lining the binding pocket. The amide bond at the end of the(CH₂)₄ aliphatic tail of biotin stacks onto PHE33 of heavy chain CDR1and is stabilized by an additional hydrogen bond to the backbone amidenitrogen of PHE33 and to Asp31. This positions the amide nitrogen, whichis the site of linkage to the active entity, in a way that atoms thatare following the nitrogen are pointing away from the binding pockettowards the solvent.

The results of the experimental determination of the binding region at aresolution of 2.5 Å enables the characterization of the binding mode ofthe ligand to its antibody, which is a prerequisite for detailedmodeling and further improvement via protein engineering of recombinantbiotin binding modules.

Example 6 Definition and Generation of Anti-Hapten Antibody withIntroduced Functionalities for Covalent Conjugation

Derivatization of the humanized VH and VL sequences of the anti-haptenantibody described above was done to generate compounds that permitcovalent coupling of antigens/haptens to the antibody at a definedposition.

The experimentally determined structure of an anti-digoxigeninFab-fragment bound to digoxigenin (3RA7) (Metz, S. et al., Proc. Natl.Acad. Sci. USA 108 (2011) 8194-8199) was used to identify positions inwhich alterations enable a site-directed coupling reaction to occurbetween the antibody and its complexed antigen/hapten. The structure ofthe anti-biotin Fab-fragment bound to biocytinamid (see Example 5) wasused to confirm the correct position of the introduced cysteine residuefor the biotin-binding antibody fragment and provide the proof of thegeneral applicability of the identified position(s).

The positions to be mutated must simultaneously meet two requirements:(i) the coupling positions should be in proximity to the binding regionto utilize the antigen/hapten positioning effect for directed coupling,and (ii) the mutation and coupling position must be positioned in amanner that antigen/hapten binding by itself is not affected. Theserequirements for finding a suitable position are de facto‘contradicting’ each other because requirement (i) is best served by aposition close to the binding site, while requirement (ii) is mostsafely achieved by positions that are distant from the binding site.

Despite these virtually excluding requirements, we were able to identifypositions that can be mutated without affecting hapten positioning, andwhich nevertheless simultaneously allow directed covalent coupling of ahaptenylated compound.

The first position is located at position VH52b or VH53 according to theKabat numbering depending on the actual length of the CDR2 of therespective antibody. In the anti-digoxigenin antibody structure, thehapten is bound in a deep pocket formed by hydrophobic residues. Afluorescent digoxigenin-Cy5 conjugate was used in this crystallographicstudy, wherein the fluorophore as well as the linker between digoxigeninand Cy5 were not visible in the structure due to a high flexibility andresulting disorder in the crystal. However, the linker and Cy5 areattached to 032 of digoxigenin which points into the direction of theCDR2 of the heavy chain. The distance between 032 (see above) ofdigoxigenin to the Cα of the amino acid residue in position 52baccording to Kabat numbering is 10.5 Å.

Replacement of the amino acid at position VH52b/VH53 with Cys generatedantibody derivatives with heavy chain variable region sequences that arelisted as SEQ ID NO: 20 and 28 for anti-digoxigenin antibody-VH52bC, SEQID NO: 84 and 92 for anti-theophylline antibody-VH53C, SEQ ID NO: 52 and60 for anti-biotin antibody-VH53C, and SEQ ID NO: 108 foranti-fluorescein antibody-VH52bC.

A further position that was identified as modification point is theposition VH28 according to the Kabat numbering.

In consequence, we introduced a cysteine at Kabat position VH28.Replacement of the amino acid at position VH28 with Cys generatedantibody derivatives with heavy chain variable region sequences that arelisted as SEQ ID NO: 124 and 132 for anti-digoxigenin antibody-VH28C,SEQ ID NO: 156 and 164 for anti-theophylline antibody-VH28C, SEQ ID NO:140 and 148 for anti-biotin antibody-VH28C, and SEQ ID NO: 116 foranti-fluorescein antibody-VH28C.

It has been found that one of these positions is a ‘universal’ position,i.e. this position is applicable to any antibody and, thus, it is notrequired to start from scratch every time a new antibody has to bemodified by providing the crystal structure and determining theappropriate position that enables hapten-positioned covalent coupling.

The mutation VH52bC or VH53C, respectively, according to Kabat heavychain variable region numbering could be used for each hapten-bindingantibody (anti-hapten antibody). Even though the antibodies andstructures of their binding pockets are quite diverse, it has been shownthat the VH52bC/VH53C mutation can be used for covalent attachment ofantigens/haptens to antibodies that bind digoxigenin, biotin,fluorescein, as well as theophylline.

Binding entities that are composed of these sequences could be expressedand purified with standard Protein A—and size exclusion chromatography(see Example 7). The resulting molecules were fully functional andretained affinity towards their cognate haptens in the same manner astheir unmodified parent molecules. This was demonstrated bySurface-Plasmon-Resonance (SPR) experiments (see Example 9).

Example 7 Composition, Expression and Purification of RecombinantAnti-Hapten Antibodies

Murine and humanized anti-hapten antibody variable regions were combinedwith constant regions of human origin to form mono- or bispecificchimeric or humanized antibodies.

The generation of monospecific humanized anti-hapten antibodies andbispecific humanized anti-hapten antibodies that specifically bind ahapten as well as a different non-hapten target (e.g. receptor tyrosinekinases or IGF-1R) required (i) design and definition of amino acid andnucleotide sequences for such molecules, (ii) expression of thesemolecules in transfected cultured mammalian cells, and (iii)purification of these molecules from the supernatants of transfectedcells. These steps were performed as previously described inPCT/EP2011/074273.

In general, to generate a humanized antibody of the IgG class that hasthe binding specificity of the (original) murine anti-hapten antibody,the humanized VH sequence was fused in frame to the N-terminus ofCH1-hinge-CH2-CH3 of a human Fc-region of the subclass IgG1. Similarly,the humanized VL sequence was fused in frame to the N-terminus of humanCLkappa constant region.

To generate bispecific antibody derivatives that contain thehapten-binding specificity as well as specificities to other targets,the anti-hapten antibody, a scFv or Fab fragment was fused in frame tothe C-terminus of the heavy chain of previously described antibodies. Inmany cases, the applied anti-hapten scFv was further stabilized byintroduction of a VH44-VL100 disulfide bond which has been previouslydescribed (e.g. Reiter, Y., et al., Nature biotechnology 14 (1996)1239-1245).

Expression Plasmids

Expression plasmids comprising expression cassettes for the expressionof the heavy and light chains were separately assembled in mammaliancell expression vectors.

Thereby the gene segments encoding the individual elements were joinedas outlined above.

General information regarding the nucleotide sequences of human lightand heavy chains from which the codon usage can be deduced is given in:Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication No 91-3242.

The transcription unit of the κ-light chain is composed of the followingelements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (hCMV),    -   a synthetic 5′-UT including a Kozak sequence,    -   a murine immunoglobulin heavy chain signal sequence including        the signal sequence intron,    -   the cloned variable light chain cDNA arranged with a unique BsmI        restriction site at the 5′ end and a splice donor site and a        unique NotI restriction site at the 3′ end,    -   the genomic human K-gene constant region, including the intron 2        mouse Ig-κ enhancer (Picard, D., and Schaffner, W. Nature        307 (1984) 80-82), and    -   the human immunoglobulin K-polyadenylation (“poly A”) signal        sequence.

The transcription unit of the γ1-heavy chain is composed of thefollowing elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (hCMV),    -   a synthetic 5′-UT including a Kozak sequence,    -   a modified murine immunoglobulin heavy chain signal sequence        including the signal sequence intron,    -   the cloned monospecific variable heavy chain cDNA or the cloned        bispecific fusion scFv-variable heavy chain cDNA arranged with a        unique BsmI restriction site at the 5′ and a splice donor site        and a unique NotI restriction site at the 3′ end,    -   the genomic human γ1-heavy gene constant region, including the        mouse Ig μ-enhancer (Neuberger, M. S., EMBO J. 2 (1983)        1373-1378), and    -   the human γ1-immunoglobulin polyadenylation (“polyA”) signal        sequence.

Beside the κ-light chain or γ1-heavy chain expression cassette theseplasmids contain

-   -   a hygromycin resistance gene,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

Recombinant DNA Techniques

Cloning was performed using standard cloning techniques as described inSambrook et al., 1999 (supra). All molecular biological reagents werecommercially available (if not indicated otherwise) and were usedaccording to the manufacturer's instructions.

DNA that contains coding sequences, mutations or further geneticelements was synthesized by Geneart AG, Regensburg.

DNA sequences were determined by double strand sequencing performed atSequiServe (SequiServe GmbH, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The Vector NTI Advance suite version 9.0 was used for sequence creation,mapping, analysis, annotation, and illustration.

Expression of Anti-Hapten Antibodies and Derivatives

The anti-hapten antibodies were expressed by transient transfection ofhuman embryonic kidney 293 (HEK293) cells in suspension. For that, lightand heavy chains of the corresponding mono- or bispecific antibodieswere constructed in expression vectors carrying prokaryotic andeukaryotic selection markers as outlined above. These plasmids wereamplified in E. coli, purified, and subsequently applied for transienttransfections. Standard cell culture techniques were used for handlingof the cells as described in Current Protocols in Cell Biology (2000),Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J.and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

The cells were cultivated in appropriate expression medium at 37° C./8%CO₂. On the day of transfection the cells were seeded in fresh medium ata density of 1-2×10⁶ viable cells/ml. The DNA-complexes withtransfection reagents were prepared in Opti-MEM I medium (Invitrogen,USA) comprising 250 μg of heavy and light chain plasmid DNA in a 1:1molar ratio for a 250 ml final transfection volume. The monospecific orbispecific antibody containing cell culture supernatants were clarified7 days after transfection by centrifugation at 14,000 g for 30 minutesand filtration through a sterile filter (0.22 μm). Supernatants werestored at −20° C. until purification.

To determine the concentration of antibodies and derivatives in the cellculture supernatants, affinity HPLC chromatography was applied. Forthat, the cell culture supernatant containing mono- or bispecificantibody or derivatives thereof that bind to protein-A was applied to anApplied Biosystems Poros A/20 column in a solution comprising 200 mMKH₂PO₄, 100 mM sodium citrate, at pH 7.4. Elution from thechromatography material was performed by applying a solution comprising200 mM NaCl, 100 mM citric acid, at pH 2.5. An UltiMate 3000 HPLC system(Dionex) was used. The eluted protein was quantified by UV absorbanceand integration of peak areas. A purified IgG1 antibody served as astandard.

Purification of Anti-Hapten Antibodies that Bind Digoxigenin,Fluorescein, Theophylline or Biotin

Seven days after transfection the HEK 293 cell supernatants wereharvested. The recombinant antibody (or -derivatives) contained thereinwere purified from the supernatant in two steps by affinitychromatography using protein A-Sepharose™ affinity chromatography (GEHealthcare, Sweden) and Superdex200 size exclusion chromatography.Briefly, the antibody containing clarified culture supernatants wereapplied on a MabSelectSuRe Protein A (5-50 ml) column equilibrated withPBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH7.4). Unbound proteins were washed out with equilibration buffer. Theantibodies (or -derivatives) were eluted with 50 mM citrate buffer, pH3.2. The protein containing fractions were neutralized with 0.1 ml 2 MTris buffer, pH 9.0. Then, the eluted protein fractions were pooled,concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K,Millipore) and loaded on a Superdex200 HiLoad 26/60 gel filtrationcolumn (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mMNaCl, at pH 6.0. The protein concentration of purified antibodies andderivatives was determined by determining the optical density (OD) at280 nm with the OD at 320 nm as the background correction, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace et. al., Protein Science 4 (1995) 2411-2423.Monomeric antibody fractions were pooled, snap-frozen and stored at −80°C. Part of the samples was provided for subsequent protein analytics andcharacterization.

The homogeneity of the antibodies was confirmed by SDS-PAGE in thepresence and absence of a reducing agent (5 mM 1,4-dithiotreitol) andstaining with Coomassie brilliant blue. The NuPAGE® Pre-Cast gel system(Invitrogen, USA) was used according to the manufacturer's instruction(4-20% Tris-Glycine gels).

Under reducing conditions, polypeptide chains related to the IgG wereidentified after SDS-PAGE at apparent molecular sizes analogous to thecalculated molecular weights. Expression levels of all constructs wereanalyzed by protein A. Average protein yields were between 6 mg and 35mg of purified protein per liter of cell-culture supernatant in suchnon-optimized transient expression experiments.

Example 8 Generation of Haptenylated Compounds

For the generation of compounds for non-covalent complexation as well asfor conjugation (covalent complexation) it is necessary (i) to couplethe hapten via suitable linkers to the compound (=payload), and (ii) toassure that the coupling occurs in a manner that allows the compound toretain its functionality.

a) Hapten-Polypeptide Conjugates:

Any polypeptide can be derivatized N- or C-terminal or in a side-chainposition by the hapten bearing linker as long as a reactive residue,such as a cysteine residue, can be introduced into the linker betweenpolypeptide and hapten. Especially the polypeptide can comprisenon-natural amino acid residues.

Exemplary haptenylated compounds are listed in the following Table 5.

TABLE 5 compound FIG. Ac-PYY(PEG3-Cys-4Abu-NH₂) 10Ac-Ile-Lys(N-propyl-(OCH₂CH₂)₃-Cys-4Abu-NH₂)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂ DIG-3-cme-eda-Cy511 DIG-maleiimid-Cy5 12 DIG-eda-Cys-Cy5 13 DIG-Ahx-Cys-Cy5 14DIG-Cys-MR121 15 Ac-PYY(PEG3-Dig) 16Ac-Ile-Lys(N-(Digoxigenin-3-carboxlmethyl-N-12-amino-4,7,10-trioxadodecanoic acid)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂ Ac-PYY(PEG3-Cys-4Abu-Dig) 17Ac-Ile-Lys(N-(Digoxigenin-3-carboxlmethyl-N-4-amino-butyricacidyl-N-Cysteinyl-N-12-amino-4,7,10-trioxododecanoic acid)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg- Tyr-NH₂PEG3-PYY(PEG3-Cys-4Abu-Dig) 18 3,6,9-trioxo-decanoicacidyl-Ile-Lys(N-propyl-(OCH₂CH₂)₃-Cys-Abu-Dig-3cme)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂ Dy636-eda-Btn 19 Dy636-Ser-Btn 20 Dy636-Cys-Btn 21Cy5-Cys-Btn 22 Cy5-Ser-Btn 23 Ac-PYY(PEG2-Btn) 24Ac-Ile-Lys(N-carboxymethyl-(OCH₂CH₂)₂—NH-Btn)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂Ac-PYY(PEG3-Cys-β-Ala-Btn) 25Ac-Ile-Lys(N-carboxymethyl-(OCH₂CH₂)₃—NH-Cys-β-Ala-Btn)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg- Tyr-NH₂Ac-PYY(PEG3-Ser-PEG2-Btn) 26Ac-Ile-Lys(N-arboxymethyl-(OCH₂CH₂)₃—NH-Ser-carboxymethyl-(OCH₂CH₂)₂-NH-Btn)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂ Ac-PYY(PEG3-Cys-PEG2-Btn) 27Ac-Ile-Lys(N-carboxymethyl-(OCH₂CH₂)₃—NH-Cys-carboxymethyl-(OCH₂CH₂)₂-NH-Btn)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂Ac-PYY(PEG3-Cys-4-Abu-5-Fluo) 28Ac-Ile-Lys(N-carboxymethyl-(OCH₂CH₂)₃—NH-Cys-4Abu-5-Fluo)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg- Tyr-NH₂Ac-PYY (PEG3-Cys-PEG2-5-Fluo) 29Ac-Ile-Lys(N-carboxymethyl-(OCH₂CH₂)₃—NH-Cys-carboxymethyl-(OCH₂CH₂)₂-NH-5-Fluo)-Pqa-Arg-His-Tyr-Leu-Asn-Trp-Val-Thr-Arg-Gln-(NMe)Arg-Tyr-NH₂ Abbreviations: 4Abu =4-Amino-butyric acid Ahx = Aminohexanoic acid Btn = biotinyl cme =carboxymethyl Cy5 = Indodicarbocyanine, Cyanin-5 Dadoo =1,8-Diamino-3,6-dioxo-octane DCM = dichloromethane Dig(OSu) =Digoxigenin-3-carboxylmethyl-N-hydroxysuccinimide Dy636 = Fluorophoreeda = ethylenediamine Fluo = 5-Carboxy-fluorescein HATU =0-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate HFIP = 1,1,1,3,3,3,-hexafluoro-2-propanol Mmt =4-Methoxytrityl MR121 = Oxazine fluorophore MTBE = tert.Butyl-methyl-ether NMM = N-Methyl-morpholine NMP =N-Methyl-2-pyrrolidone PEG2 = 8-amino-3,6-dioxa-octanoic acid PEG3 =12-amino-4,7,10-trioxadodecanoic acid O₂Oc = 8-amino-3,6-dioxa-octanoicacid Pip = piperidine Pqa =4-oxo-6-piperazin-1-yl-4H-quinazolin-3-yl)-acetic acid TBTU =2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborateTCEP = Tris(2-chloroethyl)phosphate TFE = 2,2,2,Trifluoroethanole TIS =Triisopropylsilane

A scheme of the coupling procedure and the employed reagents is shown inFIGS. 30, 31 and 32.

An exemplary polypeptide that has been used herein was a neuropeptide-2receptor agonist derivative. This polypeptide is a Peptide TyrosineTyrosine or Pancreatic Peptide YY short PYY(3-36) analog as reported inWO 2007/065808. It was digoxigenylated via the amino acid residue lysinein position 2. The digoxigenylated PYY polypeptide is termed DIG-PYY inthe following text irrespective of the side-chain linking thepolypeptide to the digoxigenin residue.

Other exemplary compounds are the non-peptide fluorescent dyes Cy5,Dy636 and MR121. These compounds can be coupled to the digoxigenin orbiotin containing linker systems via NHS-ester chemistry.

i) General Method for the Generation of the PYY(3-36)-DerivedPolypeptide Conjugation Precursor

Standard protocol for PYY derivatives on an automated multiplesynthesizer:

Synthesizer:

Multiple Synthesizer SYRO I (MultiSynTech GmbH, Witten) with vortexstirring system

Resin:

200 mg TentaGel S RAM (0.25 mmol/g), RAPP Polymere, Tubingen, 10 mlplastic syringe with a Teflon frit as reaction vessel

Stock Solutions:

Fmoc amino acids: 0.5 M in DMF or NMP

Deblocking reagent: 30% piperidine in DMF

Activator: 0.5 M TBTU and HATU, respectively

Base: 50% NMM in NMP

Coupling:

Fmoc amino acid: 519 μl

Base: 116 μl

Activator: 519 μl

Reaction time: double coupling: 2×30 min

Fmoc-Deblock:

Deblocking reagent: 1200 μl

Reaction time: 5 min+12 min

Washing:

Solvent: 1200 μl

Volume: 1300 μl

Reaction time: 5×1 min

Final Cleavage:

Cleavage reagent: 8 ml TFA/thioanisol/thiocresol/TIS (95:2, 5:2, 5:3)

Reaction time: 4 h

Work-up:

The cleavage solution was filtered and concentrated to 1-2 ml and thepeptide precipitated by addition of MTBE. The white solid was collectedby centrifugation, washed 2 times with MTBE and dried.

Ac-IK-Pqa-R(Pbf)H(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(Pbf)Q(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176)

The PYY(3-36)-polypeptide derivative (termed PYY) was obtained byautomated solid-phase synthesis of the resin-bound peptide sequenceAc-IK(Mmt)-Pqa-R(Pbf)H(Trt)Y(tBu)LN(Trt)W(Boc)VT(tBu)R(Pbf)Q(Trt)-MeArg(Mtr)-Y(tBu)-TentaGel-RAMresin. Peptide synthesis was performed according in a MultipleSynthesizer SYRO I (MultiSynTech GmbH, Witten) with vortex stiflingsystem using Fmoc chemistry. Employing a TentaGel RAM resin (loading:0.25 mmol/g; Rapp Polymers, Germany), the peptide sequence was assembledin iterative cycles by sequential coupling of the correspondingFmoc-amino acids (scale: 0.05 mmol). In every coupling step, theN-terminal Fmoc-group was removed by treatment of the resin (5 min+12min) with 30% piperidine in Dimethylformamide (DMF). Couplings werecarried out employing Fmoc-protected amino acids (0.25 mmol) activatedby TBTU (0.25 mmol) at positions 1, 13, 14 and 15 and NMM 50% in NMP(double coupling 2×30 min vortex). At all other positions HATU (0.25mmol) and NMM 50% in NMP was used as activator. Between each couplingstep the resin was washed 5×1 min with DMF. After synthesis of thelinear precursor, acetylation was performed by reaction withDMF/DIPEA/Ac₂O in 15 min and washing with DMF yieldingAc-IK(Mmt)-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin.

For the removal of the Mmt group, the peptide was treated withDCM/HFIP/TFE/TIS (6.5:2:1:0.5), 2×1 h, yielding the partial deblockedprecursorAc-IK-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin after washing with DMF.

Ac-PYY(PEG3-Dig)/Ac-IK(PEG3-Dig)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂ (SEQ ID NO:177)

Synthesis see also WO 2012/093068.

To a solution of peptide Ac-IK(H₂N-TEG)-Pqa-RHYLNWVTRQ(N-methyl)RY (100mg, 40.6 μmol) in water (5 mL) was addedDigoxigenin-3-carboxy-methyl-N-hydroxysuccinimide (26.6 mg, 48.8 μmol)dissolved in NMP (1 mL). Triethylamine (13.6 L, 97.6 μmol) was added andthe mixture was tumbled for 2 h at room temperature. Subsequently,additional Digoxigenin-3-carboxy-methyl-N-hydroxysuccinimide (13.3 mg,24.4 μmol) dissolved in NMP (0.5 mL), and triethylamine (6.8 μL, 48.8μmol) were added and the solution was tumbled for 15 h. The crudeproduct was purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) to furnish the Dig-PYY peptide (29 mg, 10.0μmol, 25%) as a colorless solid. For analytical characterization of thepeptide derivative we applied the following conditions an d received thefollowing data: Analytical HPLC: t_(R)=11.3 min (Merck ChromolithPerformance RP-18e, 100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1%TFA 80:20, 25 min); ESI-MS (positive ion mode): m/z: calcd forC₁₄₀H₂₀₇N₃₅O₃₂: 2892, 4. found: 964.9 [M+2H]²⁺, calcd: 965.1. Until thepoint of complexation to the antibody, we stored the digoxigenatedpeptide as lyophilisate at 4° C. FIG. 2C shows the structure ofDIG-moPYY.

ii) Generation of the Digoxigenylated PYY(3-36-Derived Polypeptides witha Cysteine Containing Linker

Ac-IK(PEG3-Cys-4Abu-NH₂)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂ (SEQ ID NO: 178)

Starting with the precursorAc-IK-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176) the peptide synthesis was continued withfollowing steps:

Manual double coupling with 66.5 mg (3 eq.)Fmoc-12-amino-4,7,10-trioxadodecanoic acid (PEGS-spacer), 57.0 mg (3equiv.) HATU and 16.7 μl (3 equiv.) NMM in 1.2 ml DMF for 2×30 min Afterwashings with DMF (5×1 min) the Fmoc-group was cleaved with 30% Pip/DMFand the resin was washed with DMF using the standard protocol.

The following double couplings of Fmoc-Cys(Trt)-OH and Fmoc-4-Abu-OHwere performed automatically in the SYRO 1 synthesizer by means of theprotocol as described in the standard protocol for PYY derivatives on anautomated multiple synthesizer. Finally the resin was washed with DMF,EtOH, MTBE and dried.

Cleavage from the resin was performed with 8 mlTFA/thioanisol/thiocresol/TIS (95:2.5:2.5:3) for 4 h. The cleavagesolution was filtered and concentrated to 1-2 ml and the peptideprecipitated by addition of MTBE. The white solid was collected bycentrifugation, washed 2 times with MTBE and dried.

The crude product was purified by preparative reversed phase HPLC givinga colorless solid. Yield: 28.0 mg.

Purification Protocol

HPLC: Shimadzu LC-8A with UV-Vis-detector SPD-6A

Solvent A: 0.05% TFA in water

Solvent B: 0.05% TFA in 80% acetonitrile/water

Column: UltraSep ES, RP-18, 10 μm, 250×20 mm (SEPSERV, Berlin)

Flow: 15 ml/min

Detection: 230 nm

Gradient: 20-50% B in 30 min

Analytical Data:

HPLC: Shimadzu LC-9A with photodiode array-detector SPD-M6A

Solvent A: 0.05% TFA in water

Solvent B: 0.05% TFA in 80% acetonitrile/water

Column: UltraSep ES, RP-18, 7 μm, 250×3 mm (SEPSERV, Berlin)

Flow: 0.6 ml/min

Gradient: 5-80% B in 30 min

MS: Shimadzu time-of-flight mass spectrometer AXIMA Linear (MALDI-TOF),molecular weights are calculated as average mass

m/z: calc. for C122H185N37O28S=2650.13. found: 2650.3.

Ac-IK(PEG3-Cys-4Abu-Dig)-Pqa-RHYLNWVTRQ-MeArg-Y-NH2 (SEQ ID NO: 179)

To a solution of 15 mg of peptideAc-IK(PEG3-Cys-4Abu-NH₂)-Pqa-RHYLNWVTRQ-MeArg-Y-amide (SEQ ID NO: 180)in 50 μl DMSO, 250 μl PBS buffer pH 7.4 was added and the solutionstirred overnight. The dimer formation was controlled by HPLC. After 18h app. 90% of the dimer was formed.

To this solution was added 7.3 mgDigoxigenin-3-carboxy-methyl-N-hydroxysuccinimide (Dig-OSu) dissolved in100 μl DMF and the mixture was stirred for 5 h at room temperature.Subsequently, additional 16.9 mg Dig-OSu dissolved in 100 μl DMF wasadded and stirred for 2 h. Further amount of 6.9 mg in 100 μl DMF wasadded and stirred for 18 h. For the reduction of the dimer TCEP wasadded, stirred for 3 h and the solution was used directly forpurification by means of preparative reversed phase HPLC.

Analytical Data:

Conditions were the same as described for SEQ ID NO: 178

Gradient for preparative HPLC: 38-58% B in 30 min

Yield: 5.3 mg

m/z: calc. for C₁₄₇H₂₁₉N₃₇O₃₄S=3080.7. found: 3079.8.

PEG3-IK(PEG3-Cys-4Abu-Dig)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂ (SEQ ID NO: 180)

Automated solid-phase synthesis of resin-bound PYY sequence:

(SEQ ID NO: 181) PEG2−IK(ivDde)−Pqa−R(Pbf)H(Trt)Y(tBu)LN(Trt)W(Boc)VT(tBu)R(Pbf)Q(Trt)−MeArg(Mtr)−Y(tBu)− TentaGel−RAM resin

The peptide synthesis was performed according to established protocols(FastMoc 0.25 mmol) in an automated Applied Biosystems ABI 433A peptidesynthesizer using Fmoc chemistry. Employing a TentaGel RAM resin(loading: 0.18 mmol/g; Rapp Polymers, Germany), the peptide sequence wasassembled in iterative cycles by sequential coupling of thecorresponding Fmoc-amino acids (scale: 0.25 mmol). In every couplingstep, the N-terminal Fmoc-group was removed by treatment of the resin(3×2.5 min) with 20% piperidine in N-methyl pyrrolidone (NMP). Couplingswere carried out employing Fmoc-protected amino acids (1 mmol) activatedby HBTU/HOBt (1 mmol each) and DIPEA (2 mmol) in DMF (45-60 min vortex).At positions 2, 3, and 14, respectively, the amino acid derivativesFmoc-Lys(ivDde)-OH, Fmoc-Pqa-OH, and Fmoc-N-Me-Arg(Mtr)-OH wereincorporated into the synthesis sequence. After every coupling step,unreacted amino groups were capped by treatment with a mixture of Ac₂O(0.5 M), DIPEA (0.125 M) and HOBt (0.015 M) in NMP (10 min vortex).Between each step, the resin was extensively washed with N-methylpyrrolidone and DMF. Incorporation of sterically hindered amino acidswas accomplished in automated double couplings. For this purpose, theresin was treated twice with 1 mmol of the activated building blockwithout a capping step in between coupling cycles. After completion ofthe target sequence, the N-terminal Fmoc-group was removed with 20%piperidine in NMP and 2-[2-(Methoxyethoxy)-ethoxy]acetic acid (4 mmol)was coupled after activation with HBTU/HOBt (2 mmol each) and DIPEA (4mmol). Subsequently, the resin was transferred into a frittedsolid-phase reactor for further manipulations.

PEG2-IK(PEG3-Cys-Abu-NH2)-Pqa-RHYLNWVTRQ-MeArg-Y-NH2 (SEQ ID NO: 182)

For the removal of the ivDde group, the peptide resin(PEG2-IK(ivDde)-Pqa-R(Pbf)H(Trt)Y(tBu)LN(Trt)W(Boc)VT(tBu)R(Pbf)Q(Trt)-MeArg(Mtr)-Y(tBu)-TentaGel-RAMresin; SEQ ID NO: 181) was swelled with DMF for 30 min, and wassubsequently treated with a 2% solution of hydrazine hydrate in DMF (60mL) for 2 h. After washing the resin extensively with isopropanol andDMF, a solution of Fmoc-12-amino-4,7,10-trioxadodecanoic acid(PEG3-spacer) (887 mg, 2 mmol), HBTU (2 mmol), HOBt (2 mmol) and a 2 Mdiisopropylethyl amine (2 mL, 4 mmol) in DMF (3 mL) was added, and themixture was shaken for 3 h. The resin was washed with DMF and theFmoc-group was cleaved with a mixture 20% pyridine in DMF. Subsequently,the resin was treated with a mixture of Fmoc-Cys(Trt)-OH (1.2 g; 2mmol), HBTU/HOBt (2 mmol each) and DIPEA (4 mmol) for 2 h. The resin waswashed with DMF and the Fmoc-group was cleaved with a mixture 20%pyridine in DMF and Fmoc-4-aminobutyric acid (0.65 g, 2 mmol) activatedwith HBTU/HOBt (2 mmol each) and DIPEA (4 mmol) was coupled (2 h). TheN-terminal Fmoc-group was removed with 20% piperidine in NMP and theresin washed repeatedly with DMF. Subsequently, the resin was treatedwith a mixture of trifluoroacetic acid, water and triisopropylsilane (19mL: 0.5 mL: 0.5 mL) for 2.5 h. The cleavage solution was filtered andthe peptide was precipitated by addition of cold (0° C.) diisopropylether (300 mL) to furnish a colorless solid, which was repeatedly washedwith diisopropyl ether. The crude product was re-dissolved in a mixtureof acetic acid/water and lyophilized and purified by preparativereversed phase HPLC employing an acetonitrile/water gradient containing0.1% TFA (Merck Chromolith prep RP-18e column, 100×25 mm).

Analytical HPLC: tR=8.6 min (Merck Chromolith Performance RP-18e,100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1% TFA 80:20, 25 min);ESI-MS (positive ion mode): m/z: calcd. for C127H195N37O31S: 2768.3.found: 1385.0 [M+2H]2+. calcd: 1385.1; 923.7 [M+3H]3+, calcd: 923.8;693.1 [M+4H]4+, calcd: 693.1.

PEG2-IK(PEG3-Cys-4Abu-Dig)-Pqa-RHYLNWVTRQ-MeArg-Y-NH2(PEG2-PYY(PEG3-Cys-4Abu-Dig) (SEQ ID NO: 183)

To a solution of peptidePEG2-IK(PEG3-Cys-Abu-NH2)-Pqa-RHYLNWVTRQ-MeArg-Y-NH2 (SEQ ID NO: 182,4.1 mg, 1.48 μmol) in DMF (3 mL) was addedDigoxigenin-3-carboxy-methyl-N-hydroxysuccinimide (0.81 mg, 1.48 μmol)dissolved in NMP (1 mL). Triethylamine (0.41 μl, 97.6 μmol) in DMF wasadded and the mixture was tumbled for 2 h at room temperature. The crudeproduct was purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) to furnish the PEG3-Cys-4Abu-Dig peptide (1.2mg, 0.375 μmol, 25%) as a colorless solid.

Analytical HPLC: tR=10.2 min (Merck Chromolith Performance RP-18e,100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1% TFA 80:20, 25 min);ESI-MS (positive ion mode): m/z: calcd for C152H229N37O37S: 3198.8.found: 1067.3 [M+3H]3+. calcd: 1067.3.

iii) Generation of PYY(3-36)-Derived Polypeptides with Biotin or withBiotin and Cysteine Containing Linker:

Ac-IK(PEG2-Biotin)-Pqa-RHYLNWVTRQ-MeArg-Y-Amide/Ac-PYY(PEG2-Biot) (SEQID NO: 184)

Starting with the common precursor peptide resin (SEQ ID NO: 176), thepeptide was coupled manually 2 times with 57.8 mg (3 equiv.)Fmoc-8-amino-dioxaoctanoic acid (PEG2 spacer), 48.2 mg (3 equiv.) TBTUand 33.3 μl (6 equiv.) NMM in 1.2 ml DMF, 30 min each and washed withDMF. The Fmoc-group was cleaved with 30% Pip/DMF using the standardprotocol described for SEQ ID NO: 176, the resin was washed with DMF andtreated for 2 h with a biotin-OBt solution in NMP (48.9 mg biotin (4equiv.), 64.2 mg TBTU (4 equiv.) and 44.4 μl NMM (8 equiv.) in 1.2 mlNMP, pre-activation 3 min) After washing with DMF, EtOH and MTBE thepeptide resin was dried.

Final cleavage was performed as described above. The crude product waspurified by preparative reversed phase HPLC employing a gradient of22-52% B in 30 min giving a solid. Yield: 42 mg.

Purification Protocol

HPLC: Shimadzu LC-8A with UV-Vis-detector SPD-6A

Solvent A: 0.05% TFA in water

Solvent B: 0.05% TFA in 80% acetonitrile/water

Column: UltraSep ES, RP-18, 10 μm, 250×20 mm (SEPSERV, Berlin)

Flow: 15 ml/min

Detection: 230 nm

Analytical Data:

HPLC: Shimadzu LC-9A with photodiode array-detector SPD-M6A

Solvent A: 0.05% TFA in water

Solvent B: 0.05% TFA in 80% acetonitrile/water

Column: UltraSep ES, RP-18, 7 μm, 250×3 mm (SEPSERV, Berlin)

Flow: 0.6 ml/min

Gradient: 5-80% B in 30 min

MS: Shimadzu time-of-flight mass spectrometer AXIMA Linear (MALDI-TOF),molecular weights are calculated as average mass

m/z: calc. for C₁₂₂H₁₈₁N₃₇O₂₇S=2630.10. found: 2631.5.

Ac-IK(PEG3-Cys-β-Ala-Biotin)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂/Ac-PYY(PEG3-Cys-β-Ala-Biot)(SEQ ID NO: 185)

Starting with the precursorAc-IK-Pqa-R(Pbf)H(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(Pbf)Q(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176) the peptide was coupled manually 2 times 30min with 66.5 mg (3 equiv.) Fmoc-12-amino-4,7,10-trioxadodecanoic acid(PEGS-spacer), 57.0 mg (3 equiv.) HATU and 16.7 μl (3 equiv.) NMM in 1.2ml DMF. After washing with DMF the Fmoc-group was cleaved with 30%Pip/DMF and the resin was washed with DMF using the standard protocol.

Following double couplings of Fmoc-Cys(Trt)-OH and Fmoc-β-Ala-OHperformed automatically in the SYRO 1 synthesizer by means of thestandard protocol, a solution of biotin-OBt in NMP (prepared from 48.9mg biotin (4 equiv.), 64.2 mg TBTU (4 equiv.) and 44.4 μl NMM (8 equiv.)in 1.2 ml NMP, pre-activation 3 min) was added manually and stirred atroom temperature. After 2 h the resin was washed with DMF, EtOH, MTBEand dried.

Final cleavage was performed as described above. The crude product waspurified by preparative reversed phase HPLC as described for SEQ ID NO:184 giving a colorless solid. Yield: 41.4 mg

Analytical Data:

Gradient for preparative HPLC: 28-58% B in 30 min

m/z: calc. for C₁₃₁H₁₉₇N₃₉O₃₀S₂=2862.4. found: 2862.4.

Ac-IK(PEG3-Cys-PEG2-Biotin)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂/Ac-PYY(PEG3-Cys-PEG2-Biot)(SEQ ID NO: 186)

Starting with the precursorAc-IK-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176) the peptide synthesis was continued withfollowing steps:

double coupling with Fmoc-PEG3-OH (by means of the standard protocol),

double coupling of Fmoc Cys(Trt)-OH (by means of the standard protocol),

double coupling of Fmoc-PEG2-OH with 57.8 mg (3 equiv.)Fmoc-8-amino-dioxaoctanoic acid (PEG2 spacer), 48.2 mg (3 equiv.) TBTUand 33.3 μl (6 equiv.) NMM in 1.2 ml DMF, 2×30 min and biotinylationwith a solution of 48.9 mg biotin (4 equiv.), 64.2 mg TBTU (4 equiv.)and 44.4 μl NMM (8 equiv.) in 1.2 ml NMP, (pre-activation 3 min), singlecoupling 2 h.

Cleavage from the resin, purification and analysis was performed asdescribed in for SEQ ID NO: 184. Yield: 47.7 mg

Analytical Data:

The same conditions as for SEQ ID NO: 184.

Gradient for preparative HPLC: 25-45% B in 30 min.

m/z: calc. for C₁₃₄H₂₀₃N₃₉O₃₂S₂=2936.5. found: 2937.8.

iv) Generation of PYY(3-36)-Derived Polypeptides with a Fluorescein orwith a Fluorescein and Cysteine Containing Linker

Ac-IK(PEG3-Cys-4-Abu-5-Fluo)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂/Ac-PYY(PEG3-Cys-4-Abu-5-Fluo)(SEQ ID NO: 187)

Starting with the precursorAc-IK-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176) the peptide synthesis was continuedanalogously to SEQ ID NO: 179. For labeling a solution of 54.2 mg5-Carboxyfluorescein, 33.1 mg HOBt and 35.6 μl DIC in DMF was added andstirred for 18 h at room temperature.

Cleavage from the resin, purification and analysis was performed asdescribed in for SEQ ID NO: 179. Yield: 41.6 mg

Analytical Data:

Gradient for preparative HPLC: 29-49% B in 30 min.

m/z: calc. for C₁₄₃H₁₉₅N₃₇O₃₄S=3008.44. found: 3007.2.

Ac-IK(PEG3-Cys-PEG2-5-Fluo)-Pqa-RHYLNWVTRQ-MeArg-Y-NH₂/Ac-PYY(PEG3-Cys-PEG2-5-Fluo)(SEQ ID NO: 188)

Starting with the precursorAc-IK-Pqa-R(PbeH(Trt)Y(tBu)LN(Trt)W(Boc)VT(But)R(PbeQ(Trt)-MeArg(Mtr)-Y(tBu)-TentaGelS RAM resin (SEQ ID NO: 176) the peptide synthesis was continued withfollowing steps:

double coupling with Fmoc-PEG3-OH (by means of the standard protocol),

double coupling of Fmoc Cys(Trt)-OH (by means of the standard protocol),

double coupling Fmoc-PEG2-OH (see SEQ ID NO: 186).

For the labeling the peptide resin was stirred for 18 h with a solutionof 56.7 mg 5-Carboxyfluorescein, 34.6 mg HOBt and 37.3 μl DIC in DMF.Cleavage from the resin, purification and analysis were performed asdescribed in SEQ ID NO; 185. Yield: 41.7 mg

Analytical Data:

Gradient for preparative HPLC: 34-64% B in 30 min.

m/z: calc. for C₁₄₅H₁₉₉N₃₇O₃₆S₁=3068.5. found: 3069.2.

b) Hapten-Labeled Fluorescent Dyes:

i) Generation of Digoxigenylated Cy5

Syntheses see WO 2012/093068.

ii) Generation of Dig-Cys-MR121

In an Erlenmeyer flask 1,2-Diamino-propane trityl resin (250 mg, 0.225mmol, loading 0.9 mmol/g) was swelled with DMF (5 mL) for 30 min.Subsequently, a solution of Fmoc-Cys(Trt)-OH (395 mg, 0.675 mmol) in DMF(2 mL) and a solution of HATU (433 mg, 1.2375 mmol) and HOAt (164 mg,1.2375 mmol) in DMF (8 mL) were added to the resin. To this suspensionwas added DIPEA (385 μL, 2.25 mmol) and the mixture was swirled for 16 hat ambient temperature, filtered, and washed repeatedly with DMF. Afterthe coupling step, unreacted amino groups were capped by treatment witha mixture of Ac₂O (20%) in DMF followed by a washing step with DMF.Removal of the N-terminal Fmoc group was accomplished by treatment ofthe resin with piperidine (20%) in DMF for 2 h. Afterwards, the resinwas washed thoroughly with DMF and isopropanol, and again DMF and wasthen treated with a solution of MR121 (25 mg, 0.05 mmol) in 1% DIPEA inDMF (10 mL) for 16 h. After filtration and washing with DMF, the resinwas treated with a mixture of trifluoroacetic acid, water andtriisopropylsilane (9 mL: 9 mL: 1 mL) for 3 h. The cleavage solution wasfiltered, concentrated under reduced pressure, and the resulting solidwas purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) and lyophilized. Analytical HPLC: t_(R)=7.7min (Merck Chromolith Performance RP-18e, 100×4.6 mm, water+0.1%TFA→acetonitrile/water+0.1% TFA 80:20, 25 min. Subsequently, a portionof this intermediate (10.0 mg, 17.6 μmol) was dissolved in DMF (1 mL)and a solution of Digoxigenin-3-carboxy-methyl-N-hydroxysuccinimide (9.6mg, 17.6 μmol) in DMF (1 mL) and 1% triethylamine in DMF (2 mL) wereadded and the mixture was tumbled for 16 h. The solution wasconcentrated afterwards, and the target compound was purified bypreparative reversed phase HPLC employing an acetonitrile/water gradientcontaining 0.1% TFA (Merck Chromolith prep RP-18e column, 100×25 mm)Yield: 1.0 mg. Analytical HPLC: t_(R)=10.1 min (Merck ChromolithPerformance RP-18e, 100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1%TFA 80:20, 25 min. ESI-MS (positive ion mode): m/z: calcd for [M]:996.3. found: 995.8 [M]⁺.

iii) Generation of DIG-Cys-Ahx-Cy5

In an Erlenmeyer flask 1,2-Diamino-propane trityl resin (250 mg, 0.225mmol, loading 0.9 mmol/g) was swelled with DMF (5 mL) for 30 min.Subsequently, a solution of Fmoc-Cys(Trt)-OH (395 mg, 0.675 mmol) in DMF(2 mL) and a solution of HATU (433 mg, 1.2375 mmol) and HOAt (164 mg,1.2375 mmol) in DMF (8 mL) were added to the resin. To this suspensionwas added DIPEA (385 μL, 2.25 mmol) and the mixture was swirled for 16 hat ambient temperature, filtered, and washed repeatedly with DMF. Afterthe coupling step, unreacted amino groups were capped by treatment witha mixture of Ac₂O (20%) in DMF followed by a washing step with DMF.Removal of the N-terminal Fmoc group was accomplished by treatment ofthe resin with piperidine (20%) in DMF. Afterwards, the resin was washedthoroughly with DMF and isopropanol, and again DMF and was then treatedwith a solution of Cy5-Mono NHS-ester (25 mg, 0.0316 mmol) in 1% DIPEAin DMF (10 mL) for 16 h. After filtration and washing with DMF, theresin was treated with a mixture of trifluoroacetic acid, water andtriisopropylsilane (9 mL: 9 mL: 1 mL) for 3 h. The cleavage solution wasfiltered, concentrated under reduced pressure, and the resulting solidwas re-dissolved in water and lyophilized. Purification of theintermediate was accomplished by preparative reversed phase HPLCemploying an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a blue solidafter lyophilization. Analytical HPLC: t_(R)=6.2 min (Merck ChromolithPerformance RP-18e, 100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1%TFA 80:20, 25 min. Subsequently, a portion of this intermediate (6.5 mg,7.9 μmol) was dissolved in DMF (1 mL) and a solution of Dig-Amcap-OSu(5.2 mg, 7.9 μmol) in DMF (1 mL) and 1% triethylamine in DMF (2 mL) wereadded and the mixture was tumbled for 16 h. The solution wasconcentrated afterwards, and the target compound was purified bypreparative reversed phase HPLC employing an acetonitrile/water gradientcontaining 0.1% TFA (Merck Chromolith prep RP-18e column, 100×25 mm)Yield: 3 mg. Analytical HPLC: t_(R)=8.7 min (Merck ChromolithPerformance RP-18e, 100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1%TFA 80:20, 25 min. ESI-MS (positive ion mode): m/z: calcd for [M]:1360.0. found: 1360.7 [M+H]¹⁺.

iv) Generation of Biotin-Eda-Dy636

To a solution of biotin-ethylenediamine hydrobromide (2.14 mg, 5.83μmol) in 0.1 M K₃PO₄ buffer (pH 8.0, 500 μL) was added a solution ofDy636-OSu (5 mg, 5.83 μmol) in 0.1 M K₃PO₄ buffer (pH 8.0, 500 μL) andthe resulting mixture was tumbled for 2 h at ambient temperature,filtered, and the target compound was isolated by preparative reversedphase HPLC employing an acetonitrile/water gradient containing 0.1% TFA(Merck Chromolith prep RP-18e column, 100×25 mm) After lyophilizationthe Dy636-Ethylendiamin-Bi conjugate was obtained as a colorless solid(2.8 mg, 48% %). Analytical HPLC: t_(R)=8 5 min (Merck ChromolithPerformance RP-18e, 100×4.6 mm, water+0.1% TFA→acetonitrile/water+0.1%TFA 80:20, 25 mM); ESI-MS (positive ion mode): m/z: calcd forC₅₀H₆₅N₆O₁₀S₃: 1006.3. found: 1007.3 [M+H]⁺.

v) Generation of Biotin-Ser-Dy636

Step 1: Biotin-O₂Oc-Ser-O₂Oc-DADOO—NH₂

On an O-bis-(aminoethyl)ethylene glycol trityl resin (176 mg, 0.125mmol, loading 0.71 mmol/g, Novabiochem) Fmoc-O₂Oc-OH, Fmoc-Ser(tBu)-OH,Fmoc-O₂Oc-OH (all his Biotech), and DMTr-D-Biotin (Roche) were coupledconsecutively. Peptide synthesis was performed according to establishedprotocols (FastMoc 0.25 mmol) in an automated Applied Biosystems ABI433A peptide synthesizer using Fmoc chemistry (as described for SEQ IDNO: 180).

After synthesis, the resin was washed thoroughly with DMF, methanol,dichloromethane, and dried under vacuum. Then, the resin was placed intoan Erlenmeyer flask and treated with a mixture of trifluoroacetic acid,water and triisopropylsilane (9.5 mL: 250 μL: 250 μL) for 2 h at roomtemperature. The cleavage solution was filtered and the peptide wasprecipitated by addition of cold (0° C.) diisopropyl ether (80 mL) tofurnish a colorless solid, which was repeatedly washed with diisopropylether. The crude product was re-dissolved in water, lyophilized andsubsequently purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) resulting in a colorless solid afterlyophilization. Yield: 56 mg (60%). Analytical HPLC: t_(R)=4.5 mM (MerckChromolith Performance RP-18e, 100×3 mm, water+0.1%TFA→acetonitrile/water+0.1% TFA 80:20, 25 mM ESI-MS (positive ion mode):m/z: calcd for [M]: 751.9. found: 752.4 [M+H]⁺; 376.9 [M+2H]²⁺.

Step 2: Biotin-O₂Oc-Ser-O₂Oc-DADOO-Dy-636 (Bi-Ser-Dy-636)

The peptide (5.3 mg, 7.0 μmol) was dissolved in 200 mM potassiumphosphate buffer, pH 7.5 (583 μL). Dy-636 NHS-ester (4 mg, 4.7 μmol,Dyomics) was dissolved in water (583 μL) and added to the peptidesolution. The reaction solution was stirred for 2 hours at roomtemperature and was subsequently purified by preparative reversed phaseHPLC employing an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a blue solidafter lyophilization. Yield: 3.9 mg (55%). Analytical HPLC: t_(R)=8.3 mM(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.025%TFA→acetonitrile/water+0.023% TFA 80:20, 25 min. ESI-MS (positive ionmode): m/z: calcd for [M]: 1472.8. found: 1472.8 [M+H]⁺; 737.0 [M+2H]²⁺.

vi) Generation of Biotin-Cys-Dy636

Step 1: Biotin-O₂Oc-Cys-O₂Oc-DADOO—NH₂

On an O-bis-(aminoethyl)ethylene glycol trityl resin (352 mg, 0.25 mmol,loading 0.71 mmol/g, Novabiochem) Fmoc-O₂Oc-OH, Fmoc-Cys(Trt)-OH,Fmoc-O₂Oc-OH (all his Biotech), and DMTr-D-Biotin (Roche) were coupledconsecutively. Peptide synthesis was performed according to establishedprotocols (FastMoc 0.25 mmol) in an automated Applied Biosystems ABI433A peptide synthesizer using Fmoc chemistry (as described in for SEQID NO: 180).

After synthesis, the resin was washed thoroughly with DMF, methanol,dichloromethane, and dried under vacuum. Then, the resin was placed intoan Erlenmeyer flask and treated with a mixture of trifluoroacetic acid,water and triisopropylsilane (9.5 mL: 250 μL: 250 μL) for 2 h at roomtemperature. The cleavage solution was filtered and the peptide wasprecipitated by addition of cold (0° C.) diisopropyl ether (100 mL) tofurnish a colorless solid, which was repeatedly washed with diisopropylether. The crude product was re-dissolved in water, lyophilized andsubsequently purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) resulting in a colorless solid afterlyophilization. Yield: 79 mg (41%). Analytical HPLC: t_(R)=5.3 mM (MerckChromolith Performance RP-18e, 100×3 mm, water+0.1%TFA→acetonitrile/water+0.1% TFA 80:20, 25 mM ESI-MS (positive ion mode):m/z: calcd for [M]: 767.9. found: 768.4 [M+H]⁺; 384.8 [M+2H]²⁺.

Step 2: Biotin-O₂Oc-Cys(TNB)-O₂Oc-DADOO—NH₂

The peptide (30 mg, 39 μmol) was dissolved in 100 mM potassium phosphatebuffer, pH 7.5 (4 mL) and 5,5′-dithiobis(2-nitrobenzoic acid) (77 mg,195 μmol) was added. The mixture was stirred for 30 minutes at roomtemperature and subsequently purified by preparative reversed phase HPLCemploying an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a yellow solidafter lyophilization. Yield: 31 mg (83%). Analytical HPLC: t_(R)=5 4 mM(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.025%TFA→acetonitrile/water+0.023% TFA 80:20, 25 mM ESI-MS (positive ionmode): m/z: calcd for [M]: 965.1. found: 965.4 [M+H]⁺; 483.3 [1\4+2H]²⁺.

Step 3: Biotin-O₂Oc-Cys(TNB)-O₂Oc-DADOO-Dy-636

The TNB protected peptide (1.35 mg, 1.4 μmol) was dissolved in 200 mMpotassium phosphate buffer, pH 7.5 (291 μL). Dy-636 NHS-ester (1 mg, 1.2μmol, Dyomics) was dissolved in water (291 μL) and added to the peptidesolution. The reaction solution was stirred for 1 hour at roomtemperature and was subsequently purified by preparative reversed phaseHPLC employing an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a blue solidafter lyophilization. Yield: 1 mg (50%). Analytical HPLC: t_(R)=9.0 min(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.025%TFA→acetonitrile/water+0.023% TFA 80:20, 25 min. ESI-MS (positive ionmode): m/z: calcd for [M]: 1686.0. found: 1686.7 [M+H]⁺; 844.2 [M+2H]²⁺.

Step 4: Biotin-O₂Oc-Cys-O₂Oc-DADOO-Dy-636 (Bi-Cys-Dy-636)

The TNB protected and dye labeled peptide (1 mg, 0.6 μmol) was dissolvedin a mixture of 200 mM potassium phosphate buffer, pH 7.5 (250 μL) andwater (192 μL). 100 mM tris(2-carboxyethyl)phosphine hydrochloridesolution (58 μL) was added and the reaction mixture was stirred for 30minutes at room temperature. Purification was performed by preparativereversed phase HPLC employing an acetonitrile/water gradient containing0.1% TFA (Merck Chromolith prep RP-18e column, 100×25 mm) resulting in ablue solid after lyophilization. Yield: 0.7 mg (79%). Analytical HPLC:t_(R)=8 6 mM (Merck Chromolith Performance RP-18e, 100×3 mm,water+0.025% TFA→acetonitrile/water+0.023% TFA 80:20, 25 mM ESI-MS(positive ion mode): m/z: calcd for [M]: 1488.9. found: 1488.6 [M+H]⁺;745.1 [M+2H]²⁺.

vii) Generation of Biotin-Cys-Cy5

Step 1: Biotin-O₂Oc-Cys-O₂Oc-DADOO—NH₂

On an O-bis-(aminoethyl)ethylene glycol trityl resin (352 mg, 0.25 mmol,loading 0.71 mmol/g, Novabiochem) Fmoc-O₂Oc-OH, Fmoc-Cys(Trt)-OH,Fmoc-O₂Oc-OH (all his Biotech), and DMTr-D-Biotin (Roche) were coupledconsecutively. Peptide synthesis was performed according to establishedprotocols (FastMoc 0.25 mmol) in an automated Applied Biosystems ABI433A peptide synthesizer using Fmoc chemistry (as described for SEQ IDNO: 180).

After synthesis, the resin was washed thoroughly with DMF, methanol,dichloromethane, and dried under vacuum. Then, the resin was placed intoan Erlenmeyer flask and treated with a mixture of trifluoroacetic acid,water and triisopropylsilane (9.5 mL: 250 μL: 250 μL) for 2 h at roomtemperature. The cleavage solution was filtered and the peptide wasprecipitated by addition of cold (0° C.) diisopropyl ether (100 mL) tofurnish a colorless solid, which was repeatedly washed with diisopropylether. The crude product was re-dissolved in water, lyophilized andsubsequently purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) resulting in a colorless solid afterlyophilization. Yield: 79 mg (41%). Analytical HPLC: t_(R)=5.3 min(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.1%TFA→acetonitrile/water+0.1% TFA 80:20, 25 min. ESI-MS (positive ionmode): m/z: calcd for [M]: 767.9. found: 768.4 [M+H]⁺; 384.8 [M+2H]²⁺.

Step 2: Biotin-O₂Oc-Cys(TNB)-O₂Oc-DADOO—NH₂

The peptide (30 mg, 39 μmol) was dissolved in 100 mM potassium phosphatebuffer, pH 7.5 (4 mL) and 5,5′-dithiobis(2-nitrobenzoic acid) (77 mg,195 μmol) was added. The mixture was stirred for 30 minutes at roomtemperature and subsequently purified by preparative reversed phase HPLCemploying an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a yellow solidafter lyophilization. Yield: 31 mg (83%). Analytical HPLC: t_(R)=5 4 min(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.025%TFA→acetonitrile/water+0.023% TFA 80:20, 25 min. ESI-MS (positive ionmode): m/z: calcd for [M]: 965.1. found: 965.4 [M+H]⁺; 483.3 [M+2H]²⁺.

Step 3: Biotin-O₂Oc-Cys(TNB)-O₂Oc-DADOO-Cy5

The TNB protected peptide (9.9 mg, 10.3 μmol) was dissolved in 200 mMpotassium phosphate buffer, pH 7.5 (1026 μL). Cy5-Mono NHS-ester (6.5mg, 8.2 μmol, GE Healthcare) was dissolved in water (1026 μL) and addedto the peptide solution. The reaction solution was stirred for 2 hoursat room temperature and was subsequently purified by preparativereversed phase HPLC employing an acetonitrile/water gradient containing0.1% TFA (Merck Chromolith prep RP-18e column, 100×25 mm) resulting in ablue solid after lyophilization. Yield: 10 mg (80%). Analytical HPLC:t_(R)=7.2 min (Merck Chromolith Performance RP-18e, 100×3 mm,water+0.025% TFA→acetonitrile/water+0.023% TFA 80:20, 25 mM ESI-MS(positive ion mode): m/z: calcd for [M]: 1603.9. found: 1604.9 [M+H]⁺;803.1 [M+2H]²⁺.

Step 4: Biotin-O₂Oc-Cys-O₂Oc-DADOO-Cy5 (Bi-Cys-Cy5)

The TNB protected and dye labeled peptide (10 mg, 6.1 μmol) wasdissolved in a mixture of 200 mM potassium phosphate buffer, pH 7.5(1522 μL) and water (1218 μL). 100 mM tris(2-carboxyethyl)phosphinehydrochloride solution (304 μL) was added and the reaction mixture wasstirred for 30 minutes at room temperature. Purification was performedby preparative reversed phase HPLC employing an acetonitrile/watergradient containing 0.1% TFA (Merck Chromolith prep RP-18e column,100×25 mm) resulting in a blue solid after lyophilization. Yield: 7.6 mg(86%). Analytical HPLC: t_(R)=6.4 mM (Merck Chromolith PerformanceRP-18e, 100×3 mm, water+0.025% TFA→acetonitrile/water+0.023% TFA 80:20,25 mM ESI-MS (positive ion mode): m/z: calcd for [M]: 1406.8. found:1406.8 [M+H]⁺; 704.0 [1\4+2H]²⁺.

viii) Generation of Biotin-Ser-Cy5

Step 1: Biotin-O₂Oc-Ser-O₂Oc-DADOO—NH₂

On an O-bis-(aminoethyl)ethylene glycol trityl resin (176 mg, 0.125mmol, loading 0.71 mmol/g, Novabiochem) Fmoc-O₂Oc-OH, Fmoc-Ser(tBu)-OH,Fmoc-O₂Oc-OH (all his Biotech), and DMTr-D-Biotin (Roche) were coupledconsecutively. Peptide synthesis was performed according to establishedprotocols (FastMoc 0.25 mmol) in an automated Applied Biosystems ABI433A peptide synthesizer using Fmoc chemistry (as described for SEQ IDNO: 180).

After synthesis, the resin was washed thoroughly with DMF, methanol,dichloromethane, and dried under vacuum. Then, the resin was placed intoan Erlenmeyer flask and treated with a mixture of trifluoroacetic acid,water and triisopropylsilane (9.5 mL: 250 μL: 250 μL) for 2 h at roomtemperature. The cleavage solution was filtered and the peptide wasprecipitated by addition of cold (0° C.) diisopropyl ether (80 mL) tofurnish a colorless solid, which was repeatedly washed with diisopropylether. The crude product was re-dissolved in water, lyophilized andsubsequently purified by preparative reversed phase HPLC employing anacetonitrile/water gradient containing 0.1% TFA (Merck Chromolith prepRP-18e column, 100×25 mm) resulting in a colorless solid afterlyophilization. Yield: 56 mg (60%). Analytical HPLC: t_(R)=4.5 mM (MerckChromolith Performance RP-18e, 100×3 mm, water+0.1%TFA→acetonitrile/water+0.1% TFA 80:20, 25 mM ESI-MS (positive ion mode):m/z: calcd for [M]: 751.9. found: 752.4 [M+H]⁺; 376.9 [M+2H]²⁺.

Step 2: Biotin-O₂Oc-Ser-O₂Oc-DADOO-Cy5 (Bi-Ser-Cy5)

The peptide (5.7 mg, 7.6 μmol) was dissolved in 200 mM potassiumphosphate buffer, pH 7.5 (789 μL). Cy5-Mono NHS-ester (5 mg, 6.3 μmol,GE Healthcare) was dissolved in water (789 μL) and added to the peptidesolution. The reaction solution was stirred for 2 hours at roomtemperature and was subsequently purified by preparative reversed phaseHPLC employing an acetonitrile/water gradient containing 0.1% TFA (MerckChromolith prep RP-18e column, 100×25 mm) resulting in a blue solidafter lyophilization. Yield: 6 mg (58%). Analytical HPLC: t_(R)=6.1 mM(Merck Chromolith Performance RP-18e, 100×3 mm, water+0.025%TFA→acetonitrile/water+0.023% TFA 80:20, 25 mM ESI-MS (positive ionmode): m/z: calcd for [M]: 1390.72. found: 1391.2 [M+H]⁺.

Example 9 Binding of Recombinant Humanized Anti-Biotin Antibody toBiotin-Labeled Compound (Haptenylated Compound)

In order to determine whether the humanization procedure and thesubsequent introduction of cysteine mutations resulted in derivativesthat had retained full binding activity the following experiments wereperformed.

The binding properties of the recombinant anti-biotin antibodyderivatives were analyzed by biolayer interferometry (BLI) technologyusing an Octet QK instrument (Fortebio Inc.). This system is wellestablished for the study of molecule interactions. BLi-technology isbased on the measurement of the interference pattern of white lightreflected from the surface of a biosensor tip and an internal reference.Binding of molecules to the biosensor tip is resulting in a shift of theinterference pattern which can be measured. To analyze if thehumanization procedure described above diminished the ability of theanti-biotin antibody to bind to biotin, the properties of the chimericand the humanized versions of the antibody in their ability to bind to abiotinylated protein were compared directly. Binding studies wereperformed by capturing anti-biotin antibody on anti-huIgG Fc antibodyCapture (AHC) Biosensors (Fortebio Inc.). First, biosensors wereincubated in an antibody solution with a concentration of 0.5 mg/ml in20 mM histidine, 140 mM NaCl, pH 6.0 for 1 min. Thereafter, thebiosensors were incubated for 1 mM in 1×PBS pH 7.4 to reach a stablebaseline. Binding was measured by incubating the antibody-coatedbiosensors in a solution containing biotinylated protein with aconcentration of 0.06 mg/ml in 20 mM histidine, 140 mM NaCl, pH 6.0 for5 mM Dissociation was monitored for 5 min in 1×PBS pH 7.4. The resultingbinding curves for chimeric and humanized anti-biotin antibodies werecompared directly.

The humanized version of the antibody showed equal or even betterbinding of the biotinylated antigen than the chimeric antibody. The sameis true for the humanized antibody with the Cys mutation at Kabatposition VH53. The biotinylated protein showed residual unspecificbinding to the biosensors which was reduced when the biosensors werecoated with Herceptin, which does not bind biotin. Thus, thefunctionality of the anti-biotin antibody was retained in its humanizedvariant (which is defined by the sequences as depicted in SEQ ID NO: 44and 48, SEQ ID NO: 60 and 64).

Surface Plasmon Resonance

Surface plasmon resonance measurement was performed on a BIAcore® T200instrument (GE Healthcare Biosciences AB, Sweden) at 25° C. Around 4300resonance units (RU) of the capturing system (10 μg/ml Anti-humanCapture (IgG Fc) from Human Antibody Capture Kit, BR-1008-39, GEHealthcare Biosciences AB, Sweden) were coupled on a CM3 chip (GEHealthcare, BR-1005-36) at pH 5.0 by using the standard amine couplingkit supplied by GE Healthcare (BR-1000-50). The running buffer for aminecoupling was HBS-N (10 mM HEPES, pH 7.4, 150 mM NaCl, GE Healthcare,BR-1006-70). Running and dilution buffer for the followed binding studywas PBS-T (10 mM phosphate buffered saline including 0.05% Tween 20) pH7.4. The humanized anti-biotin antibody was captured by injecting a 2 nMsolution for 60 sec at a flow rate of 5 μl/min. Biotinylated siRNA wasdiluted with PBS-T at concentrations of 0.14-100 nM (1:3 dilutionseries). Binding was measured by injecting each concentration for 180sec at a flow rate of 30 μl/min, dissociation time 600 sec. The surfacewas regenerated by 30 sec washing with a 3 M MgCl₂ solution at a flowrate of 5 μl/min. The data were evaluated using BIAevaluation software(GE Healthcare Biosciences AB, Sweden). Bulk refractive indexdifferences were corrected by subtracting the response obtained from ananti-human IgG Fc surface. Blank injections were also subtracted(=double referencing). For calculation of KD and kinetic parameters theLangmuir 1:1 model was used.

Kinetic binding analysis by surface plasmon resonance (SPR) was carriedout for humanized anti-biotin antibody SEQ ID NO: 44 and 48 andhumanized anti-biotin antibody VH53C SEQ ID NO: 60 and 64. Anti-biotinantibodies at a concentration of 2 nM were captured by anti-human IgG Fcantibody which was bound to a CM3 sensor chip. Binding of biotinylatedsiRNA (Mw: 13868 Da) was recorded at the concentrations 0.41, 1.23, 3.7,11.1, 33.3, 100 and 300 nM. Measurements were carried out in duplicates.The calculated K_(D) for humanized anti-biotin antibody and humanizedanti-biotin antibody VH53C were 0.633 nM and 0.654 nM, respectively.

Example 10 Generation of Non-Covalent Complexes of HaptenylatedCompounds with Anti-Hapten Antibodies

General Method:

The generation of complexes of anti-hapten antibodies with haptenylatedcompounds (=haptens conjugated to a payload) shall result in definedcomplexes and it shall be assure that the compound (=payload) in thesecomplexes retains its activity. For the generation of complexes ofhaptenylated compounds with the respective anti-hapten antibody thehaptenylated compound was dissolved in H₂O to a final concentration of 1mg/ml. The antibody was concentrated to a final concentration of 1 mg/ml(4.85 μM) in 20 mM histidine buffer, 140 mM NaCl, pH=6.0. Haptenylatedpayload and antibody were mixed to a 2:1 molar ratio (compound toantibody) by pipetting up and down and incubated for 15 minutes at RT.

Alternatively, the haptenylated compound was dissolved in 100% DMF to afinal concentration of 10 mg/ml. The antibody was concentrated to afinal concentration of 10 mg/ml in 50 mM Tris-HCl, 1 mM EDTA, pH=8.2.Haptenylated compound and antibody were mixed to a 2.5:1 molar ratio(compound to antibody) by pipetting up and down and incubated for 60minutes at RT and 350 rpm.

Exemplary Method for the Formation of Complexes of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Non-Covalent Digoxigenin-Cy5Complex

Humanized and murine anti-digoxigenin antibody or bispecificanti-digoxigenin antibody derivatives were used as antibody components.For the generation of complexes of digoxigenylated Cy5 with theanti-digoxigenin antibodies the Cy5-digoxigenin conjugate was dissolvedin PBS to a final concentration of 0.5 mg/ml. The antibody was used in aconcentration of 1 mg/ml (about 5 μM) in a buffer composed of 20 mMhistidine and 140 mM NaCl, pH 6. Digoxigenylated Cy5 and antibody weremixed at a 2:1 molar ratio (digoxigenylated Cy5 to antibody). Thisprocedure resulted in a homogenous preparation of complexes of definedcomposition.

The complexation reaction can be monitored by determining thefluorescence (650/667 nm) of the antibody-associated fluorophore on asize exclusion column. The results of these experiments demonstrate thatcomplexation only occurs if the antibody contains binding specificitiesfor digoxigenin. Antibodies without binding specificities fordigoxigenin do not bind the digoxigenin-Cy5 conjugate. An increasingsignal can be observed for bivalent anti-digoxigenin antibodies until adigoxigenin-Cy5 conjugate to antibody ratio of 2:1. Thereafter, thecomposition dependent fluorescence signals reach a plateau.

Exemplary Method for the Formation of Complexes of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Biotin-Cy5/ChimericAnti-Biotin Antibody (Human IgG Subclass) Complex

For the generation of complexes of biotin-derivatized-Cy5(Biotin-Cys-Cy5) containing a cysteinylated linker, 0.16 mg ofBiotin-Cys-Cy5 were dissolved in 100% DMF to a concentration of 10mg/ml. 1 mg of the antibody was used in a concentration of 10.1 mg/ml(about 69 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2.Biotin-Cys-Cy5 and antibody were mixed at a 2.5:1 molar ratio(Biotin-Cys-Cy5 to antibody) and incubated for 60 mM at RT, shaken at350 rpm. The resulting conjugate was analyzed by SDS-PAGE as describedin Example 11a. Detection of fluorescence was carried out as describedin Example 11a.

Exemplary Method for the Formation of Conjugates of BiotinylatedFluorescent Dyes and Anti-Biotin Antibodies—Biotin-Ser-Cy5/HumanizedAnti-Biotin Antibody:

For the generation of complexes of biotin-derivatized-Cy5(Biotin-Ser-Cy5) containing a serine residue within the linker, 0.61 mgof Biotin-Ser-Cy5 were dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0to a concentration of 10 mg/ml. 18.5 mg of the humanized anti-biotinantibody was used in a concentration of 10 mg/ml (about 69 μM) in abuffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Ser-Cy5 andantibody were mixed at a 2.5:1 molar ratio (Biotin-Ser-Cy5 to antibody)and incubated for 60 mM at RT, shaken at 350 rpm. The sample was thensubjected to size exclusion chromatography using Superdex 200 16/60 highload prep grade column (GE Healthcare) with a flow rate of 1.5 ml/minand 20 mM histidine, 140 mM NaCl, pH 6.0 as the mobile phase. Peakfractions were collected and analyzed by SDS-PAGE for purity. The dye toantibody ratio was calculated by (1) measuring the absorbance of thesamples at the wavelength 280 nm (protein) and 650 nm (Cy5); (2) usingthe formula: A₆₅₀ of labeled protein/ϵ(Cy5)*protein concentration(M)=moles dye per mole protein, where ϵ(Cy5)=250000 M⁻¹cm⁻¹, A₆₅₀ of thecomplex=47.0 and the protein concentration is 86.67 μM. The resultingratio of dye to antibody molecule was 2.17 which suggests that allantibody paratopes are saturated with Biotin-Cy5 molecules.

Exemplary Method for the Formation of Complexes of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Digoxigenin-PYY(3-36)/Anti-Digoxigenin Antibody Complex

For the generation of non-covalent complexes of digoxigenylatedpolypeptides with an anti-digoxigenin antibody the murinehybridoma-derived antibody (lyophilisate from 10 mM KPO₄, 70 mM NaCl; pH7.5) was dissolved in 12 ml water and dialyzed against a solutioncomprising 20 mM histidine, 140 mM NaCl, pH 6.0 to yield 300 mg (2×10⁻⁶mol) in 11 ml buffer (c=27.3 mg/ml). Digoxigenin-PYY(3-36) conjugate(11.57 mg, 4×10⁻⁶ mol, 2 eq.) was added in 4 portions of 2.85 mg within1 h and incubated for another hour at room temperature. After completionof the complexation reaction, the complexes were purified by sizeexclusion chromatography via a Superdex 200 26/60 GL column (320 ml) in20 mM histidine, 140 mM NaCl, at pH 6.0 at a flow rate of 2.5 ml/min.The eluted complex was collected in 4 ml fractions, pooled andsterilized over a 0.2 μm filter to give 234 mg of the complex at aconcentration of 14.3 mg/ml. In a similar manner, for generation ofcomplexes of the humanized anti-digoxigenin antibody the antibody wasadjusted to a concentration of 10.6 mg/ml (9.81 mg, 6.5×10⁻⁸ mol in 0.93ml) in 20 mM histidine, 140 mM NaCl, pH 6.0. 0.57 mg=1.97×10⁻⁷ mol=3.03eq. of the digoxigenylated polypeptide (DIG-PYY) were added to theantibody solution as lyophilisate. Polypeptide and antibody wereincubated for 1.5 hrs. at room temperature. The excess of polypeptidewas removed by size exclusion chromatography via a Superose 6 10/300 GLcolumn in 20 mM histidine, 140 mM NaCl, at pH 6.0 at a flow rate of 0.5ml/min. The eluted complex was collected in 0.5 ml fractions, pooled andsterilized over a 0.2 μm filter to give 4.7 mg of the complex at aconcentration of 1.86 mg/ml.

The resulting haptenylated polypeptide-anti-hapten antibody complex wasdefined as monomeric IgG-like molecule via the occurrence of a singlepeak in a size exclusion chromatography. The resulting complex wasdefined as monomeric IgG-like molecule, carrying two Digoxigenin-PYYderivatives per antibody molecule. The defined composition of thesepeptide complexes was confirmed by size exclusion chromatography, whichalso indicated the absence of protein aggregates. The definedcomposition (and 2:1 polypeptide to protein ratio) of these bispecificpeptide complexes was further confirmed by SEC-MALS (Size exclusionchromatography-Multi Angle Light Scattering). For SEC-MALS analysis,100-500 μg of the respective sample was applied to a Superdex 200 10/300GL size exclusion column with a flow rate of 0.25-0.5 ml/min with 1×PBSpH 7.4 as mobile phase. Light scattering was detected with a WyattminiDawn TREOS/QELS detector, the refractive index was measured with aWyatt Optilab rEX-detector. Resulting data was analyzed using thesoftware ASTRA (version 5.3.4.14). The results of SEC MALLS analysesprovide information about the mass, radius and size of the complex.These data were then compared with those of the correspondingnon-complexed antibody. The results of these experiments demonstratethat exposure of Digoxigenylated-PYY to the anti-digoxigenin antibodyresults in complexes that contain two Digoxigenin-PYY derivatives perone antibody molecule. Thus, digoxigenylated PYY can be complexed withthe anti-digoxigenin antibody at defined sites (binding region) and witha defined stoichiometry.

Characterization of the complex by surface plasmon resonance studiesprovided additional evidence that the complexation reaction generateddefined and completely complexed molecules. The anti-digoxigeninantibody can be bound to the SPR chip which results in signal increases.Subsequent addition of digoxigenin-PYY conjugate results in furthersignal increases until all binding sites are completely occupied. Atthese conditions, addition of more Digoxigenin-PYY does not increase thesignal further. This indicates that the complexing reaction is specificand that the signals are not caused by non-specific stickiness of thedigoxigenylated polypeptide.

Exemplary Method for the Formation of Complexes of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY-PEG3-Cys-β-Ala-Biot/Chimeric Anti-Biotin AntibodyComplex

For the generation of non-covalent complexes ofbiotinylated-PYY-polypeptide containing a cysteinylated linker, 0.19 mgof Ac-PYY-PEG3-Cys-β-Ala-Biot were dissolved in 100% DMF to aconcentration of 10 mg/ml. The antibody was used in a concentration of10.7 mg/ml (about 73 μM) in a buffer composed of 50 mM Tris-HCl, 1 mMEDTA, pH 8.2. Ac-PYY-PEG3-Cys-β-Ala-Biot and antibody were mixed at a2.5:1 molar ratio (Ac-PYY-PEG3-Cys-β-Ala-Biot to antibody) and incubatedfor 60 mM at RT and 350 rpm. The resulting complex was defined asmonomeric IgG-like molecule via the occurrence of a single peak in asize exclusion chromatography (95% monomer). The resulting complex wasfurther analyzed by SDS-PAGE and subsequent Western Blot analysis. 10 μgof the complex were mixed with 4×LDS sample buffer (Invitrogen) andincubated at 95° C. for 5 mM The sample was applied to a 4-12% Bis-Trispolyacrylamide-gel (NuPAGE, Invitrogen) which was run for 35 mM at 200Vand 120 mA. Molecules that were separated in the polyacrylamide-gel weretransferred to a PVDF membrane (0.2 μm pore size, Invitrogen) for 40 mMat 25V and 160 mA. The membrane was blocked in 1% (w/v) skim milk in1×PBST (1×PBS+0.1% Tween20) for 1 h at RT. The membrane was washed 3×for 5 mM in 1×PBST and subsequently incubated with astreptavidin-POD-conjugate (2900 U/ml, Roche) which was used in a 1:2000dilution. Detection of streptavidin-POD bound to biotin on the membranewas carried out using Lumi-Light Western Blotting Substrate (Roche).

Exemplary Method for the Formation of Complexes of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY-PEG3-Cys-PEG2-Biot)/Chimeric Anti-Biotin AntibodyComplex

For the generation of non-covalent complexes ofbiotinylated-PYY-polypeptide containing a cysteinylated linker, 0.16 mgof Ac-PYY-PEG3-Cys-PEG2-Biot were dissolved in 100% DMF to aconcentration of 10 mg/ml. The antibody was used in a concentration of10.7 mg/ml (about 73 μM) in a buffer composed of 50 mM Tris-HCl, 1 mMEDTA, pH 8.2. Ac-PYY-PEG3-Cys-PEG2-Biot and antibody were mixed at a2.5:1 molar ratio (Ac-PYY-PEG3-Cys-PEG2-Biot to antibody) and incubatedfor 60 mM at RT and 350 rpm. The resulting complex was defined as 63%monomeric IgG-like molecule and 37% dimeric soluble aggregates via sizeexclusion chromatography. The resulting complex was further analyzed bySDS-PAGE and subsequent Western Blot analysis. 10 μg of the complex weremixed with 4×LDS sample buffer (Invitrogen) and incubated at 95° C. for5 mM The sample was applied to a 4-12% Bis-Tris polyacrylamide-gel(NuPAGE, Invitrogen) which was run for 35 mM at 200V and 120 mA.Molecules that were separated in the polyacrylamide-gel were transferredto a PVDF membrane (0.2 μm pore size, Invitrogen) for 40 mM at 25V and160 mA. The membrane was blocked in 1% (w/v) skim milk in 1×PBST(1×PBS+0.1% Tween20) for 1 h at RT. The membrane was washed 3× for 5 mMin 1×PBST and subsequently incubated with a streptavidin-POD-conjugate(2900 U/ml, Roche) which was used in a 1:2000 dilution. Detection ofstreptavidin-POD bound to biotin on the membrane was carried out usingLumi-Light Western Blotting Substrate (Roche).

Exemplary Method for the Formation of Complexes of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-PEG2-5-Fluo)/Chimeric Anti-FluoresceinAntibody Complex

For the generation of non-covalent complexes offluorescein-conjugated-PYY-polypeptide containing a cysteinylatedlinker, 0.33 mg of Ac-PYY(PEG3-Cys-PEG2-5-Fluo were dissolved in 100%DMF to a concentration of 10 mg/ml. The antibody was used in aconcentration of 9.99 mg/ml (about 68 μM) in a buffer composed of 50 mMTris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY(PEG3-Cys-PEG2-5-Fluo and antibodywere mixed at a 2.5:1 molar ratio (Ac-PYY(PEG3-Cys-PEG2-5-Fluo) toantibody) and incubated for 60 min at RT and 350 rpm. The resultingcomplex was defined as 76% monomeric IgG-like molecule and 24% dimericsoluble aggregates via size exclusion chromatography. The resultingcomplex was further analyzed by SDS-PAGE and subsequent detection offluorescein-related fluorescence in the polyacrylamide-gel. 8 μg of thecomplex were mixed with 4×LDS sample buffer (Invitrogen) and incubatedat 95° C. for 5 mM Fluorescein-related fluorescence was recorded using aLumiImager F1 device (Roche) at an excitation wavelength of 645 nm.

Example 11 Generation of Defined Covalent Conjugates of HaptenylatedDyes or Polypeptides with an Anti-Hapten Antibody VH52bC/VH53C in thePresence of Redox Agents

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-HaptenAntibodies—Dig-Cys-Ahx-Cy5/Anti-Digoxigenin Antibody VH52bC

The generation of covalent conjugates of anti-hapten antibodies andhaptenylated fluorescent dyes containing a cysteine-linker results indefined conjugates where a disulfide bridge is formed at a specificposition between VH52bC in the CDR2 of the anti-hapten antibody and thecysteine in the linker between the hapten and the fluorescent dye. Theconjugation reaction was carried out in the presence of redox reagents.Dig-Cys-Ahx-Cy5 was dissolved in 20 mM histidine, 140 mM NaCl, pH 6.0.Solubilization was facilitated by drop wise addition of 10% (v/v) aceticacid. The final concentration was adjusted to 0.4 mg/ml. Theanti-digoxigenin antibody VH52bC in 20 mM histidine, 140 mM NaCl, pH 6.0was brought to a concentration of 10 mg/ml. An anti-digoxigenin antibodywas used as a control and was treated the same way as anti-digoxigeninantibody VH52bC. 4.7 nmol of each antibody was mixed with 2.5 molarequivalents of Dig-Cys-Ahx-Cy5. This was achieved by adding 11.7 nmol ofthis substance in 4 portions (2.9 nmol each) every 15 mM In betweenthese additions, the samples were incubated at 25° C. while gentlyshaking. After addition of the last portion, 0.64 nmol of eachantibody-Dig-Cys-Ahx-Cy5 complex was transferred to buffer containingthe following redox reagents: 3 mM DTE (Dithioerythritol)+10 mM GSSG(oxidized Glutathione), 0.3 mM DTE+1 mM GSSG and 0.03 mM DTE+0.1 mMGSSG. All samples were incubated for 15 mM in these conditions. Afterthe incubation, samples were split into half (0.34 nmol each) andprepared for SDS gel electrophoresis. For this, 4×LDS sample buffer(Invitrogen) was added. For each sample also a reduced version wasprepared by adding 10× NuPAGE sample reducing agent (Invitrogen). Allsamples were incubated at 70° C. for 5 mM before electrophoresis on a4-12% Bis-Tris polyacrylamide gel (NuPAGE, Invitrogen) with 1×MOPSbuffer (Invitrogen). Cy5-related fluorescence in the gel was detectedwith a LumiImager F1 device (Roche) at an excitation wavelength of 645nm. After detection of fluorescence, the gel was stained with SimplyBlueSafeStain (Invitrogen). Gels are shown in FIG. 8.

Site-specific disulfide bond formation was shown for anti-digoxigeninantibody VH52bC (FIG. 8, gels on top, lanes 1 A-C) with a low backgroundfluorescence signal when anti-digoxigenin antibody without a cysteine inCDR2 was used (lanes 2 A-C). The background signals in the controlreactions can be explained by coupling of Dig-Cys-Ahx-Cy5 to cysteinesthat are normally involved in the formation of antibody-interchaindisulfide bonds. Increasing amounts of redox reagents substantiallyreduce disulfide bridges that connect antibody heavy and light chains,producing mainly ¾ antibodies (−1×LC), HC-dimers (−2×LC) and ½antibodies (1×HC+1×LC). On the bottom of the gel fluorescence ofDig-Cys-Ahx-Cy5 that was not covalently linked to the antibody can bedetected. The gels on the bottom of FIG. 8 show, that upon reduction ofthe samples, no Cy5-related fluorescence is detectable near the antibodyheavy and light chains, indicating that the covalent linkage was indeedformed by a disulfide bridge. Coomassie stains of each gel show that thetotal amount of protein in each lane was equal.

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Dig-Cys-Cy5/Anti-DigoxigeninAntibody VH52bC

Dig-Cys-Cy5 was dissolved in 8.3 mM HCl, 10% (v/v) DMF to a finalconcentration of 3.25 mg/ml. The anti-digoxigenin antibody VH52bCantibody in 20 mM histidine, 140 mM NaCl, pH 6.0 was brought to aconcentration of 15 mg/ml. anti-digoxigenin antibody was used as acontrol and was treated the same way as anti-digoxigenin antibodyVH52bC. 13.3 nmol of each antibody was mixed with 2 molar equivalents ofDig-Cys-Cy5 at a final antibody concentration of 10 mg/ml in thepresence of 1 mM GSH (reduced glutathione) and 5 mM GSSG (reducedglutathione). This was achieved by adding 26.6 nmol of this substance in2 portions every 5 mM In between these additions, the samples wereincubated at RT while gently stirred. After addition of the lastportion, the samples were incubated for 1 h at RT. The efficiency of thecoupling reaction was evaluated by SDS-PAGE and subsequent recording ofthe Cy5-related fluorescence signal. 5, 10 and 20 μg of each sample wereprepared for SDS-PAGE. For this, 4×LDS sample buffer (Invitrogen) wasadded. All samples were incubated at 70° C. for 5 min beforeelectrophoresis on a 4-12% Bis-Tris polyacrylamide gel (NuPAGE,Invitrogen) with 1×MOPS buffer (Invitrogen). Cy5-related fluorescence inthe gel was detected with a LumiImager F1 device (Roche) at anexcitation wavelength of 645 nm. After detection of fluorescence, thegel was stained with SimplyBlue SafeStain (Invitrogen).

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—PEG3-PYY(PEG3-Cys-4Abu-Dig)/Humanized Anti-DigoxigeninAntibody VH52bC

For the generation of conjugates ofdigoxigenin-derivatized-PYY-polypeptide containing a cysteinylatedlinker, 1.4 mg of PEG3-PYY(PEG3-Cys-4Abu-Dig) were dissolved in 100% DMFto a concentration of 10 mg/ml. 1 mg of the antibody was used in aconcentration of 10 mg/ml (about 68 μM) in a buffer composed of 5 mMTris-HCl, 1 mM EDTA, 1 mM GSH, 5 mM GSSG, pH 8.2.PEG3-PYY(PEG3-Cys-4Abu-Dig) and antibody were mixed at a 2:1 molar ratio(PEG3-PYY(PEG3-Cys-4Abu-Dig) to antibody) and incubated for 60 min atRT, stirred at 100 rpm. The resulting conjugate was analyzed by massspectrometry. 43% of the detected species was identified as antibodycoupled to 2 polypeptide molecules, 46% was antibody coupled to 1polypeptide molecule and 11% was identified as uncoupled antibody.

Example 12 Generation of Defined Covalent Conjugates of HaptenylatedDyes and Polypeptides with an Anti-Hapten Antibody VH52bC/NH53C in theAbsence of Redox Agents

For the generation of covalent anti-hapten antibody/haptenylatedpolypeptide or haptenylated dye disulfide-linked conjugates it isnecessary to (i) couple the hapten (e.g. digoxigenin, fluorescein,biotin or theophylline) via a suitable a reactive group (such as e.g.cysteine, maleimide) containing linkers to the polypeptide or dye thatallows the polypeptide to be exposed above the antibody surface andhence to retain its activity, and (ii) generate covalent site specificconjugates of the haptenylated polypeptides with the anti-haptenantibody with a cysteine mutation (=antibody VH52bC/VH53C) in which thebiological activity of the polypeptide is retained, and (iii) to carryout the reaction in the absence of a reducing agent in order to avoidthe reduction of antibody inter-chain disulfide bridges.

General Method:

The generation of conjugates of anti-hapten antibodies with haptenylatedcompounds shall result in conjugates with defined stoichiometry and itshall be assured that the compound in these conjugates retains itsactivity. For the generation of conjugates of haptenylated compoundswith the respective anti-hapten antibody the haptenylated compound wasdissolved in 100% DMF to a final concentration of 10 mg/ml. Theanti-hapten antibody VH52bC/VH53C was brought to a concentration of 10mg/ml in 50 mM Tris-HCl, 1 mM EDTA, pH=8.2. Haptenylated compound andanti-hapten antibody VH52bC/VH53C were mixed in a 2.5:1 molar ratio(compound to antibody) by pipetting up and down and incubated for 60minutes at RT and 350 rpm.

A polypeptide conjugated to the hapten via a cysteine containing linkeris termed hapten-Cys-polypeptide or polypeptide-Cys-hapten in thefollowing. The polypeptide may either have a free N-terminus or a cappedN-terminus e.g. with an acetyl-group (Ac-polypeptide-Cys-hapten) or aPEG-residue (PEG-polypeptide-Cys-hapten).

A fluorescent dye conjugated to the hapten via a cysteine containinglinker is termed dye-Cys-hapten or hapten-Cys-dye in the following.

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-HaptenAntibodies—Dig-Cys-Ahx-Cy5/Anti-Digoxigenin Antibody VH52bC

Samples were prepared exactly as described in Example 11a, with thedifference that antibody-Dig-Cys-Ahx-Cy5 complexes were transferred tobuffer containing either no redox compounds, 0.1 mM GSSG (oxidizedglutathione) or 1 mM GSSG. The resulting fluorescence-scanned andCoomassie stained polyacrylamide gels are shown in FIG. 9. An threeconditions show a similar specificity for site-specific disulfide bondformation (FIG. 9, top gels, lanes 1 A-C) with a low level of backgroundreactions (FIG. 9, lanes 2 A-C). This confirms that formation of thedisulfide bond can be accomplished without the need of reducing agents.This significantly stabilizes the antibody/reduces antibodydisintegration, as only residual amounts of ¾ antibodies (−1×LC),HC-dimers (−2×LC) and ½ antibodies (1×HC+1×LC) are detected incomparison to Example 11.

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Dig-Cys-Cy5/Anti-DigoxigeninAntibody VH52bC

Samples were prepared exactly as described in Example 11b, with thedifference that 13.3 nmol of antibody was mixed with 2 molar equivalentsof Dig-Cys-Cy5 at a final antibody concentration of 10 mg/ml in theabsence of redox reagents.

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Biotin-Cys-Cy5/ChimericAnti-Biotin Antibody VH53C

For the generation of conjugates of biotin-derivatized-Cy5 containing acysteinylated linker, 0.16 mg of Biotin-Cys-Cy5 were dissolved in 100%DMF to a concentration of 10 mg/ml. 1 mg of the anti-biotin antibodyVH53C was used in a concentration of 9.7 mg/ml (about 68 μM) in a buffercomposed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5 andantibody were mixed at a 2.5:1 molar ratio (Ac-Biotin-Cys-Cy5 toantibody) and incubated for 60 mM at RT, shaken at 350 rpm. Theresulting conjugate was analyzed by SDS-PAGE as described in Example11a. Detection of fluorescence was carried out as described in Example11a.

Exemplary Method for the Formation of Conjugates of HaptenylatedFluorescent Dyes and Anti-Hapten Antibodies—Biotin-Cys-Cy5/HumanizedAnti-Biotin Antibody VH53C

For the generation of conjugates of biotin-derivatized-Cy5 containing acysteinylated linker, 0.16 mg of Biotin-Cys-Cy5 were dissolved in 100%DMF to a concentration of 10 mg/ml. 1 mg of the humanized anti-biotinantibody VH53C was used in a concentration of 7.4 mg/ml (about 51 μM) ina buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2. Biotin-Cys-Cy5and antibody were mixed at a 2.5:1 molar ratio (Ac-Biotin-Cys-Cy5 toantibody) and incubated for 60 min at RT, shaken at 350 rpm. Theresulting conjugate was analyzed by SDS-PAGE as described in Example11a. Detection of fluorescence was carried out as described in Example11a.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-4Abu-Dig)/Humanized Anti-Digoxigenin AntibodyVH52bC

For the generation of conjugates ofdigoxigenin-derivatized-PYY-polypeptide containing a cysteinylatedlinker, 2.4 mg of Ac-PYY(PEG3-Cys-4Abu-Dig) were dissolved in 20%acetate to a concentration of 5 mg/ml. 10 mg of the humanizedanti-digoxigenin antibody VH52bC (68.4 nmol) was used in a concentrationof 19.5 mg/ml (about 133 μM) in a buffer composed of 20 mM histidine,140 mM NaCl, pH 6.0. Ac-PYY(PEG3-Cys-4Abu-Dig) and antibody were mixedat a 2:1 molar ratio (Ac-PYY(PEG3-Cys-4Abu-Dig) to antibody) andincubated for 60 min at RT, stirred at 100 rpm. The resulting conjugatewas analyzed by mass spectrometry. 7.4% of the detected species wasidentified as antibody coupled to 2 peptide molecules, 40% was antibodycoupled to 1 peptide molecule and 52% was identified as uncoupledantibody.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-βAla-Biot)/Chimeric Anti-Biotin AntibodyVH53C

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.19 mg of Ac-PYY(PEG3-Cys-βAla-Biot)were dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of thechimeric anti-biotin antibody VH53C was used in a concentration of 9.7mg/ml (about 67 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Ac-PYY[PEG3-Cys-βAla-Biot and antibody were mixed at a 2.5:1molar ratio (Ac-PYY[PEG3-Cys-βAla-Biot] to antibody) and incubated for60 mM at RT, shaken at 350 rpm. The resulting conjugate was analyzed bymass spectrometry. 87.7% of the detected species was identified asantibody coupled to 2 peptide molecules, 12.3% was identified asantibody coupled to 1 peptide molecule.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-PEG2-Biot)/Chimeric Anti-Biotin AntibodyVH53C

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.16 mg of Ac-PYY(PEG3-Cys-PEG2-Biot)were dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of thechimeric anti-biotin antibody VH53C was used in a concentration of 9.9mg/ml (about 68 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Ac-PYY[PEG3-Cys-PEG2-Biot and antibody were mixed at a 2.5:1molar ratio (Ac-PYY[PEG3-Cys-PEG2-Biot] to antibody) and incubated for60 mM at RT, shaken at 350 rpm. The resulting conjugate was analyzed bymass spectrometry. 100% of the detected species was identified asantibody coupled to 2 peptide molecules.

Exemplary Method for the Formation of Conjugates of Haptenylated PolyPeptides and Anti-Hapten Antibodies—Ac-PYY(PEG3-Cys-βAla-Biot)/HumanizedAnti-Biotin Antibody VH53C

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.06 mg of Ac-PYY(PEG3-Cys-βAla-Biot)were dissolved in 100% DMF to a concentration of 10 mg/ml. 0.8 mg of thehumanized anti-biotin antibody VH53C was used in a concentration of 9mg/ml (about 62 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Ac-PYY[PEG3-Cys-βAla-Biot and antibody were mixed at a 2.5:1molar ratio (Ac-PYY[PEG3-Cys-βAla-Biot] to antibody) and incubated for60 mM at RT, shaken at 350 rpm. The resulting conjugate was analyzed bymass spectrometry. 62.2% of the detected species was identified asantibody coupled to 2 peptide molecules, 33.9% was identified asantibody coupled to 1 peptide molecule and 3.9% was identified asuncoupled antibody.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-PEG2-Biot)/Humanized Anti-Biotin AntibodyVH53C

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.08 mg of Ac-PYY(PEG3-Cys-PEG2-Biot)were dissolved in 100% DMF to a concentration of 10 mg/ml. 0.8 mg of thehumanized anti-biotin antibody VH53C was used in a concentration of 9mg/ml (about 62 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Ac-PYY[PEG3-Cys-PEG2-Biot and antibody were mixed at a 2.5:1molar ratio (Ac-PYY[PEG3-Cys-PEG2-Biot] to antibody) and incubated for60 mM at RT, shaken at 350 rpm. The resulting conjugate was analyzed bymass spectrometry. 71.4% of the detected species was identified asantibody coupled to 2 peptide molecules, 26% was identified as antibodycoupled to 1 peptide molecule and 2.5% was identified as uncoupledantibody.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-PEG2-Fluo)/Anti-Fluorescein Antibody VH52bC

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.33 mg of Ac-PYY[PEG3-Cys-PEG2-Fluowere dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of theanti-fluorescein antibody VH52bC was used in a concentration of 9.3mg/ml (about 63 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Ac-PYY[PEG3-Cys-PEG2-Fluo and antibody were mixed at a 2.5:1molar ratio (Ac-PYY[PEG3-Cys-PEG2-Fluo] to antibody) and incubated for60 mM at RT, shaken at 350 rpm. The resulting conjugate was analyzed bymass spectrometry. 95% of the detected species was identified asantibody coupled to 2 peptide molecules, 5% was identified as antibodycoupled to 1 peptide molecule.

Exemplary Method for the Formation of Conjugates of HaptenylatedPolypeptides and Anti-HaptenAntibodies—Ac-PYY(PEG3-Cys-PEG2-Fluo)/Anti-Fluorescein Antibody VH28C

For the generation of conjugates of biotin-derivatized-PYY-polypeptidecontaining a cysteinylated linker, 0.33 mg of Ac-PYY[PEG3-Cys-PEG2-Fluowere dissolved in 100% DMF to a concentration of 10 mg/ml. 1 mg of theanti-fluorescein antibody VH28C was used in a concentration of 9.5 mg/ml(about 63 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2.Ac-PYY[PEG3-Cys-PEG2-Fluo and antibody were mixed at a 2.5:1 molar ratio(Ac-PYY[PEG3-Cys-PEG2-Fluo] to antibody) and incubated for 60 mM at RT,shaken at 350 rpm. The resulting conjugate was analyzed by massspectrometry. 100% of the detected species was identified as antibodycoupled to two peptide molecules.

Example 13 Generation of Covalent Theophylline-Anti-TheophyllineAntibody Complexes

To evaluate the formation of covalent antibody complexes that utilizetheophylline and theophylline-binding antibodies as hapten recognitionsystem, Theophyllin-Cys-Cy5 was generated as fluorescent payload,applying generally the synthesis and purification technologies that havebeen described for Digoxigenin-Cys-Cy5 or Biotin-Cys-Cy5, with theexception that the hapten has been exchanged against theophylline (seeExample 8 and FIGS. 13, 14 and 22). The composition of theTheophylline-Cys-Cy5 derivative that had been synthesized is shown inFIG. 43a ). To demonstrate the formation of a covalent disulfide,theophylline-binding antibodies were generated which contained adesigned Cys at position 54 or 55 of the heavy chain variable region(anti-theophylline antibody-Cys). The purity of these antibodies isshown exemplarily for the Y54C variant in FIG. 43b ). These antibodyderivatives were complexed with Theophylline-Cys-Cy5 and subsequentlysubjected to SDS-PAGE under non-reducing and reducing conditions asdescribed in Example 12. Under non-reducing conditions, disulfide-linkedanti-theophylline-antibody complexed Cy5 was detected by its H-chainassociated fluorescence within the gel in the same manner as describedin Example 12. This is depicted in FIG. 43c ), which demonstrates thatcovalent complexes between antibody had been formed as a consequence ofthe simple loading reaction in the same manner as the disulfides thatwere observed when using Digoxigenin, Fluorescein or Biotin as hapten.These complexes dissociated as expected upon reduction, i.e. releasedthe payload from the H-chain only when the disulfide became reduced(FIG. 43c )).

Example 14 Generation of Covalent Hapten-Antibody Complexes UnderIn-Vivo Like Conditions, and Evidence for Directed Disulfide-FormationIn Vivo

To evaluate the formation of covalent hapten-antibody complexes underin-vivo like conditions, anti-Biotin antibodies-Cys were incubated at37° C. in murine serum with Biotin-Cys-Cy5 for 60 min. Subsequently, theantibody was captured from the murine serum by protein-A. Thereafter thecaptured antibodies were subjected to SDS-PAGE under non-reducing andreducing conditions as described in Example 12. Disulfide-linkedantibody-complexed Cy5 was detected by its H-chain associatedfluorescence within the gel in the same manner as described in Example12. FIG. 44 demonstrates that covalent complexes between antibody formin serum at 37° C., i.e. under conditions that resemble the in-vivoconditions. These complexes dissociate as expected upon reduction, i.e.the payload is released from the H-chain only when the disulfide becomesreduced (FIG. 44). The observation that upon hapten-positioning adirected disulfide bond between antibody and payload can be formed evenin the presence of serum is unexpected as serum contains a high amountof proteins, peptides and other compounds (which can interfere withdisulfide-formation reactions). The observation that uponhapten-positioning a directed disulfide bond between antibody andpayload can be formed in serum at 37° C. also opens the possibility toapply this PK-modulation system in a pre-targeting setting: separateapplication of antibody and hapten-payload, followed by in-vivo assemblyof antibody complexes and subsequent disulfide formation.

To further evaluate potential in vivo ‘pre-targeting’ applications, thepharmacokinetics of Biotin-Cy5 was determined under pre-targetingconditions by the non-invasive optical imaging technology of the eye ofanimals as described in Example 18. In these experiments, the presenceof Cy5 was determined non-invasive by optical imaging of the eye ofanimals, which revealed the fluorescence of Cy5 in the capillaries. TheCy5-mediated fluorescence values that we detected in the eye of mice 10min. after injection of Biotin-Cy5 were set as 100% value, andfluorescence values measured at subsequent time points were expressedrelative thereto. In this experiment, 1 mg antibody (either anti-Biotinantibody or anti-Biotin antibody-Cys (=Cys-mutant of anti-Biotinantibody)) was applied 24 hours before injection of Biotin-Cy5 and startof the eye imaging. The control group was not pre-injected with theanti-biotin antibody.

The results of these experiments are shown in FIG. 45: injection ofBiotin-Cy5 into animals that did not receive pre-injected antibody waseliminated with a low serum half-life and low exposure levels(diamonds). The serum levels and half-life of Biotin-Cy5 that wasinjected into animals with 24 hours pre-injection of anti-Biotinantibody (without cys mutation) were greatly increased. This shows thatthe antibody captures its antigen (with the payload) in the circulation,and prolongs the antigen's (and likewise of the conjugated payload)serum half-life. The relative serum level and half-life ofBiotin-Cys-Cy5 that was injected into animals that were 24 hourspre-injected with the anti-Biotin antibody-cys (i.e. an antibodycontaining the cys mutation as reported herein for covalent payloadcoupling) were even further increased. In these samples, the relativeCy5 levels were not only higher than those of non-complexed compound,but also higher than the levels of complexed (but not disulfide-bonded)Cy5. Thus, hapten-complexed disulfide-linked payloads (which are formedunder pre-targeting conditions in vivo) are more stable in thecirculation, and can reach higher exposure levels, than non-covalentcomplexed payloads.

Example 15 Polypeptides in Conjugates and in Complexes with Anti-HaptenAntibody Retain Functionality

We have previously shown that polypeptides which are part ofnon-covalent hapten-polypeptide conjugates and in complexes withanti-hapten antibodies retain functionality (WO2011/003557, WO2011/003780 and PCT/EP2011/074273). To demonstrate that coupled peptidesretain functionality also upon covalent disulfide-coupling, thebiological activity of anti-digoxigenin antibody complexed polypeptidesand their disulfide-conjugates with anti-digoxigenin antibody VH52bCwere compared.

The therapeutically desired functionality of PYY-derived peptides isbinding to and interfering with the signaling of its cognate receptorNPY2. Signaling via the NPY2 receptor is involved in and/or regulatesmetabolic processes.

To evaluate whether complexation or SS-conjugation of the polypeptideDig-PYY with the anti-digoxigenin antibody or the conjugation of thepolypeptide Dig-Cys-PYY with the anti-digoxigenin antibody VH52bC,respectively, affect its activity, we evaluated its ability to inhibitthe Forskolin stimulated cAMP accumulation in HEK293 cells expressingthe NPY₂ receptor (cAMP assay).

The following Table 6 shows the results of cAMP-assays that wereperformed to assess the biological activity of PYY(3-36), its Y2receptorspecific modified analog moPYY, its antibody-complexed Dig-variant andits disulfide-conjugated Dig-Cys-derivative.

TABLE 6 day 1 day 2 sample EC₅₀ [nM] EC₅₀ [nM] PYY_(wt) 0.09 0.1 moPYY0.14 0.15 moPYY(Cys-Dig)-disulfide conjugated-anti- 5.38 5.33digoxigenin antibody VH52bC moPYY(Dig) - anti-digoxigenin antibody 9.2612.55 complex

For the cAMP agonist assay, the following materials were used: 384-wellplate; Tropix cAMP-Screen Kit; cAMP ELISA System (Applied Biosystems,cat. #T1505; CS 20000); Forskolin (Calbiochem cat. #344270); cells:HEK293/hNPY2R; growth medium: Dulbecco's modified eagle medium (D-MEM,Gibco); 10% Fetal bovine serum (FBS, Gibco), heat-inactivated; 1%Penicillin/Streptomycin (Pen 10000 unit/mL: Strep 10000 mg/mL, Gibco);500 μg/mL G418 (Geneticin, Gibco cat. #11811-031); and plating medium:DMEM/F12 w/o phenol red (Gibco); 10% FBS (Gibco, cat. #10082-147),heat-inactivated; 1% Penicillin/Streptomycin (Gibco, cat. #15140-122);500 μg/mL G418 (Geneticin, Gibco, cat. #11811-031).

To perform the assay, on the first day, medium was discarded, and themonolayer cells were washed with 10 mL PBS per flask (T225). Afterdecanting with PBS, 5 mL VERSENE (Gibco, cat #1504006) was used todislodge the cells (5 min @ 37° C.). The flask was gently tapped and thecell suspension was pooled. Each flask was rinsed with 10 mL platingmedium and centrifuged at 1000 rpm for 5 min. The suspension was pooledand counted. The suspension was resuspended in plating medium at adensity of 2.0×10⁵ cells/mL for HEK293/hNPY2R. 50 microliters of cells(HEK293/hNPY2R—10,000 cells/well) were transferred into the 384-wellplate using Multi-drop dispenser. The plates were incubated at 37° C.overnight. On the second day, the cells were checked for 75-85%confluence. The media and reagents were allowed to come to roomtemperature. Before the dilutions were prepared, the stock solution ofstimulating compound in dimethyl sulphoxide (DMSO, Sigma, cat #D2650)was allowed to warm up to 32° C. for 5-10 min. The dilutions wereprepared in DMEM/F12 with 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX,Calbiochem, cat #410957) and 0.5 mg/mL BSA. The final DMSO concentrationin the stimulation medium was 1.1% with Forskolin concentration of 5 μM.The cell medium was tapped off with a gentle inversion of the cell plateon a paper towel. 50 μl of stimulation medium was placed per well (eachconcentration done in four replicates). The plates were incubated atroom temperature for 30 min, and the cells were checked under amicroscope for toxicity. After 30 mM of treatment, the stimulation mediawas discarded and 50 μL/well of Assay Lysis Buffer (provided in theTropix kit) was added. The plates were incubated for 45 mM @ 37° C. 20μl of the lysate was transferred from stimulation plates into thepre-coated antibody plates (384-well) from the Tropix kit. 10 μl of APconjugate and 20 μl of anti-cAMP antibody were added. The plates wereincubated at room temperature while shaking for 1 hour. The plates werethen washed 5 times with Wash Buffer, 70 μl per well for each wash. Theplates were tapped to dry. 30 μl/well of CSPD/Sapphire-II RTUsubstrate/enhancer solution was added and incubated for 45 min @ RT(shake). Signal for 1 sec/well in a Luminometer. (VICTOR-V) wasmeasured.

The results of these assays (Table 6) show that the modified peptidederivative moPYY has a neglectable lower activity than the wild-typePYY. The IC₅₀ value of the cAMP assay was 0.09 nM for the wild-type PYYand 0.14 nM for the modified analog. Covalent disulfide-conjugationresulted to a slight reduction in biological activity. The IC₅₀ valuewas 5-36 nM for the conjugate. Surprisingly the covalentdisulfide-conjugate is 2-fold more active than the non-covalent complexwith an IC₅₀ value of 10.91 nM.

Example 16 Serum Stability of Complexes of Biotinylated Cy5 withHumanized Anti-Biotin Antibody in Comparison to Covalent Conjugates ofBiotinylated Cy5 with Humanized Anti-Biotin Antibody VH53C

The objective of the described peptide modification technology is toimprove the therapeutic applicability of peptides. Major bottlenecks fortherapeutic application of peptides are currently limited stability invivo and/or short serum half-life and fast clearance. The PK parametersof antibody conjugates of fluorophores were determined in vivo andcompare with the PK of non-covalent antibody-fluorophore complexes.Therefore, (i) the anti-biotin antibody VH53C was covalently conjugatedto the biotinylated fluorophore Biot-Cys-Cy5, (ii) a non-covalentcomplex of the anti-biotin antibody with biotinylated fluorophoreBiot-Cy5 was generated, (iii) the covalently conjugated and thenon-covalently complexed compounds were applied to animals and (iv) theserum concentrations of the compounds over time in these animals wasmeasured by determination of the fluorescence of Cy5 (A650), and that ofthe corresponding antibody by an ELISA method that specifically detectsthe humanized antibody.

Experimental Procedure

To analyze the influence on PK parameters of antibody-complexation of asmall fluorescent substrate, 13 nmol of Cy5-biotin/humanized anti-biotinantibody VH53C-conjugate, or of the corresponding antibodynon-covalently complexed compound, or of the fluorescent compound alone,in 20 mM histidine/140 mM NaCl, pH 6.0 were applied to six female mice(strain NRMI) for each substance. About 0.1 ml blood samples werecollected after the following time points: 0.08 h, 4 h and 48 h forMouse 1, 2, and 3 in a first group, and 0.08 h, 24 h and 72 h for Mouse1, 2 and 3 in a second group. Serum samples of at least 50 μl wereobtained after 1 h at RT by centrifugation (9300×g, 3 min, 4° C.). Serumsamples were stored at −80° C.

To determine the amount of compound in the serum at the given timepoints the fluorescent properties of Cy5 are used: Cy5 relatedfluorescence in serum samples were measured in 120 μl quartz cuvettes atroom temperature using a Cary Eclipse Fluorescence Spectrophotometer(Varian). Excitation wavelength was 640 nm, Emission was measured at 667nm. Serum samples were diluted in 1×PBS to reach an appropriate range ofEmission intensity. Blood serum of an untreated mouse in the samedilution in 1×PBS as the respective sample was used as a blank probe anddid not show any fluorescence signal.

FIG. 34 shows the results of an analysis employing covalent conjugates,non-covalent complexes and non-complexed hapten-Cy5. The data is shownas relative (%) levels of Cy5-mediated fluorescence normalized to the(peak) serum levels 5 min after injection. For a compound of rathersmall molecular weight, non-complexed Biotin-Ser-Cy5 disappears rapidlyfrom the serum. One hour after injection, only 6% of the fluorescencethat was applied and detectable after 5 minutes in the serum was stilldetectable. At later time points, 2 hrs., 4 hrs. and 24 hrs. afterinjection, Cy5-mediated signals were not detectable.

Of the antibody-complexed compound four hours after injection, stillapprox. 50% of the fluorescence that was applied (5 min levels set to100%) was detectable in the serum. Cy5-mediated fluorescence levels werealso detectable at later time points with approx. 22% of the 5 minvalues detectable at 2 hrs. and approx. 12% detectable 48 hrs. afterinjection and 8% still detectable after 72 hrs. The antibody-conjugatedcompound shows a significantly longer in vivo half-life than theantibody-complexed compound. Four hours after injection 58% of thefluorescence that was applied (5 min levels set to 100%) was stilldetectable in the serum (a factor of 1.16 higher than for theantibody-complexed compound). After 24 hrs. 35% (factor 1.6), after 48hrs. 31% (factor 2.6) and after 72 hrs. 26% (factor 3.3) of theCy5-mediated fluorescence was detected in serum. The comparable decreaseof fluorescence for complexed and conjugated compounds in the first 24hrs. of the experiments can be accounted for the early distributionwhich is similar for complexes and conjugates. After 24 hrs. the in vivostability of antibody-conjugated compounds is responsible for thedifference.

To determine the amount of human IgG antibody in the serum at the giventime points, the following assay principle was used: human IgG1antibodies in serum samples were captured on a solid phase (Maxisorb®microtiter plate, NUNC-Immuno™) coated with an anti-human kappa-chainmonoclonal IgG antibody. Serum samples were diluted 1:10⁵ and 1:10⁶ and100 μl of these dilutions were added to the wells. After incubation,wells were washed 3-times with 300 μl PBS/0.05% Tween 20 each. Detectionof human IgG antibodies was carried out by first adding 100 μl ofanti-human C_(H)1-domain IgG which is digoxigenylated at the C-terminusat a concentration of 0.25 μg/ml. After washing 3-times with 300 μl of1×PBS/0.05% Tween 20 each, 100 μl of anti-digoxigenin antibodyFab-fragment conjugated to horse-radish peroxidase (HRP) was added at aconcentration of 25 mU/ml. Finally, per well 100 μl of ABTS® were added.After 30 mM incubation at ambient temperature, the extinction (OD) wasmeasured at 405 nm and 492 nm [405/492] in a commercial microtiter plateELISA Reader (e.g. Tecan Sunrise).

FIG. 34 shows the Bio-Cy5 serum levels as well as the serum levels ofhuman IgG in mice treated with antibody-biotin-Cy5-complexes and-conjugates. The data is shown as relative (%) human IgG levelsnormalized to the (peak) serum levels 5 mM after injection. The relativehuman IgG serum levels of both antibody-hapten-complexes and -conjugatesare in-line with the relative fluorescence measured for theantibody-hapten conjugates. Thus, the Biotin-Cys-Cy5 compound shows asimilar in vivo stability as the antibody it is conjugated to, whichmeans that antibody-hapten conjugates stay intact in vivo. This isclearly not the case for antibody-hapten complexes for which therelative Cy5-mediated fluorescence decreases faster than the relativehuman IgG serum levels. This means that the complexes release thepayload over time in vivo.

In summary, the in vivo stability of haptenylated compounds issignificantly increased when bound by an anti-hapten antibody. However,antibody-hapten complexes are not completely stable in vivo as thedecrease of the hapten-Cy5 serum levels is faster than the decrease ofantibody serum levels. This is not the case for antibody-hapten-Cy5conjugates, which show a similar in vivo behavior as normal IgGantibodies.

Dig-Peptide Serum Kinetic (Comparison of Non-Covalent Complex andCovalent Conjugate)

To analyze the influence on PK parameters of antibody-complexation andantibody conjugation of the digoxigenylated polypeptide, 32.1 nmol ofthe polypeptide, or of the corresponding antibody non-covalentlycomplexed polypeptide in 20 mM histidine/140 mM NaCl pH 6.0 were appliedto 2 female mice (strain NRMI) for each substance. The mice had a weightof 23 g and 25 g for MAK-DIG-PYY and 28 g and 26 g for DIG-PYY. About0.1 ml blood samples were collected after the following time points:0.08 h, 2 h and 24 h for Mouse 1 and 0.08 h, 4 h 24 h for Mouse 2. Serumsamples of at least 40 μl were obtained after 1 h at RT bycentrifugation (9300×g, 3 mM, 4° C.). Serum samples were stored at −80°C.

The determination of the amount of digoxigenylated peptide in the serumat the given time points was difficult compared to the detection ofDig-Cy5 as no direct means to detect the polypeptide in serum sampleswas available. Therefore, a Western-Blot related assay to detectdigoxigenylated peptide in serum was established. In a first step, theserum samples were separated on reducing SDS-PAGE. Because samplepreparation included exposure of the serum to high concentrations of SDSand reducing agents, complexed Dig-polypeptide conjugates can becomereleased from the (completely denatured/unfolded) anti-digoxigeninantibody, whereas covalently conjugates remained bound. To mediate therelease of the polypeptide from the non-covalent antibody complex andseparate the individual components by SDS-PAGE, 2 μl of each serumsample was diluted in 18 μl 20 mM histidine/140 mM NaCl pH 6.0, mixedwith 6.7 μl of 4×LDS sample buffer and 3 μl of 10× sample reducing agent(NuPAGE, Invitrogen) for 5 mM at 95° C. As a control, 2 μl of serum ofan untreated mouse of the same strain was used. Samples were applied toa 4-12% Bis-Tris Gel (NuPAGE, Invitrogen) which was run at 200 V/120 mAfor 20 minutes using 1×MES (Invitrogen) as a running buffer.Subsequently, separated polypeptides were blotted onto a PVDF membrane(0.22 μm pore size, Invitrogen) using the XCell Sure Lock® Mini-Cellsystem (Invitrogen) for 40 min at 25 V/130 mA. Membranes were blocked in1% skim milk in 1×PBS+1% Tween20 (PBST) for 1 h at RT. Digoxigenylatedpolypeptides were subsequently detected on the membrane with ananti-digoxigenin antibody. For that, anti-digoxigenin antibody wasapplied to the membranes in a concentration of 13 μg/ml in 10 ml of 1%skim milk/PBST for 2 h at RT. Membranes were washed for 3×5 mM in1×PBST. Anti-mouse IgG Fab-fragments coupled to POD from theLumiLight^(PLUS) Western Blotting Kit (Roche) was applied in a 1:25dilution in 10 ml of 1% skim milk/PBST for 1 h at RT. Membranes werewashed 3×5 mM with 1×PBST. Detection was carried out by incubating themembranes in 4 ml LumiLight Western Blotting substrate for 5 min at RT.Chemiluminescence was detected with the LumiImager F1 (Roche) with anexposure time of 20 min.

The results of these analyses are shown in FIGS. 35 A and B. Thepresence/amount of the digoxigenin polypeptide in murine serum atdifferent time points has been determined. Mice that had receivedantibody complexed peptides (FIG. 35 left) showed strong signals at theearliest time point (5 mM after administration). These signals wereclearly assignable as shown by the size and location on the blot of thecontrols. In sera of mice that were treated with antibody-complexedpolypeptide, polypeptide-associated signals were strongest at the earlytime points and decreased over time. Nevertheless, polypeptide was stilldetectable with good signals at all time points and even 24 hrs. afteradministration.

In mice that received non-complexed polypeptide, barely any signalassociable to the small polypeptide was detectable even at the earliesttime point. FIG. 35 shows in the right that under normal exposureconditions, no free polypeptide is visible on the blot. Contrastenhancement of the blot revealed the presence of some polypeptide 5 minafter administration, however only in trace amounts. At later timepoints, no defined polypeptide band was detectable.

It can be seen that non-complexed polypeptide has a very short half-lifein the serum of mice. Mice that received the same polypeptides but inantibody complexed form, show presence of these polypeptides in theserum for an increased period of time. Twenty four hrs. after injectionpolypeptide can be determined in the serum of these mice.

Example 17 Serum Half-Life of Covalently Linked Digoxigenin-AntibodyComplexes and Digoxigenin-Binding IgGs

To analyze if the covalent complexation further improves thePK-properties in view of the non-covalently linked hapten complexes, thePK parameters of anti-digoxigenin antibody-Digoxigenin-Cy5 complexes, aswell as of the covalently linked [anti-digoxigeninantibody-Cys]-[Digoxigenin-Cys-Cy5] conjugates were determined in vivo.Therefore, Digoxigenin-Cy5 was determined using its fluorescence (A650),and the corresponding antibody was determined by an ELISA method thatspecifically detects the humanized antibody. Digoxigenin-Cy5 was appliedas low molecular weight ‘surrogate’ for hapten-coupled peptides becauseits fluorescent properties allow easy and accurate detection in theserum.

In the same manner as described for Biotin-Cy5 or Biotin-Cys-Cy5 (seeExample 16), Digoxigenin-Cy5 or antibody-complexed or additionallyantibody-cys-linked Digoxigenin-Cy5 were injected intravenously intofemale NRMI mice, followed by collection of blood at 0.08 h, 2 h, 4 hand 24 h. The Cy5-mediated fluorescence values detected for/in both mice5 min. after injection (t=0.08 hrs.) was set as 100% value andfluorescence values measured at subsequent time points were expressedrelative thereto.

The results of these experiments demonstrate that for Digoxigenin-Cy5less than 10% of the fluorescence that was applied (5 min value) wasdetectable 2 hours after injection. At later time points, 4 hrs. and 24hrs., respectively, after injection no Cy5-mediated signals weredetectable (see FIG. 41). In contrast to non-complexed compound,antibody-complexed compound was detectable at much higher levels and atlater time points (FIG. 41). This indicates that antibody complexationsignificantly increases the serum half-life of the small compoundDigoxigenin-Cy5. Furthermore, covalently linked payloads display agreater PK prolongation compared to the non-covalently linked complexes.A direct comparison of the Digoxigenin-Cy5 levels and antibody levelsindicated payload loss from the antibody over time, with Cy5 levelsdecreasing faster than antibody levels. In contrast, covalently linkedDigoxigenin-conjugates showed almost identical Cy5 and IgG serumhalf-lives (FIG. 41). This indicates that the disulfide-inked payloadsremain stably connected to the antibodies while the non-covalentcomplexes dissociate over time.

Example 18 Serum Half-Life and Exposure Levels of Covalently LinkedHapten-Antibody Complexes and Complexes which are Only Attached Via theHapten-Binding Site

To analyze if the covalent complexation improves the PK-properties ofnon-covalently linked hapten complexes, the PK of a complex ofanti-biotin antibody with Biotin-Cy5, as well as that of the covalentlylinked conjugate [anti-biotin-antibody-Cys]-[Biotin-Cys-Cy5] in vivowere determined. The presence of Cy5 was determined non-invasive byoptical imaging of the eye of animals, which revealed the fluorescenceof Cy5 in the capillaries. The Cy5-mediated fluorescence values that wedetected in the eye of mice 10 min after injection was set as 100%value, and fluorescence values measured at subsequent time points wereexpressed relative thereto. The results of these experiments are shownin FIG. 42: non-complexed Biotin-Cy5 by itself has a low serum half-lifeand low exposure levels. Antibody-complexed compound which was notcovalently linked was detectable at much higher levels and with anextended half-life. Furthermore, covalently linked payloads displayed agreater PK prolongation, and higher serum levels compared to thenon-covalently linked complexes. This indicates that hapten-complexeddisulfide-linked payloads are more stable in the circulation, and canreach higher exposure levels, than non-covalent complexed payloads.

Example 19 Peptide-Complexation and Covalent Conjugation with Antibodiesthat Bind Different Haptens

The application of hapten binding modules to couple haptenylatedcompounds (=payloads) to targeting vehicles is one technical possibilityby which hapten-mediated delivery can be realized. The concept can beexpanded to further haptens or other entities that capture compounds andconnect them to the targeting module. For example, for polypeptidedelivery or stabilization, mono- or bispecific antibodies that binddigoxigenin or other haptens can be applied to stabilize and PK-optimizetherapeutic polypeptides.

Prerequisites for application as polypeptide capturing modules are (i)that coupling of compounds to the hapten does not severely interferewith polypeptide activity and (ii) the possibility of effectivebinding/complexation of the antibody to haptenylated compounds.

Hapten-directed binding is a prerequisite for the efficient covalentcoupling of haptenylated dyes or polypeptides with an anti-haptencysteinylated antibody.

To show that affinity-driven complexation of haptenylated compounds withanti-hapten antibodies is a prerequisite for efficient disulfide-bondformation, Biotin-Cys-Cy5 was incubated with humanized anti-digoxigeninantibody and humanized anti-digoxigenin antibody VH53C. Incubation ofBiotin-Cys-Cy5 with humanized anti-biotin antibody and humanizedanti-biotin antibody VH53C was carried out as a control reaction.

0.13 mg of Biotin-Cys-Cy5 were dissolved in 100% DMF to a concentrationof 10 mg/ml. 0.7 mg of each antibody was used in a concentration of 6.7mg/ml (about 46 μM) in a buffer composed of 50 mM Tris-HCl, 1 mM EDTA,pH 8.2. Biotin-Cys-Cy5 and antibodies were mixed at a 2.5:1 molar ratio(Ac-Biotin-Cys-Cy5 to antibody) and incubated for 60 mM at RT, shaken at350 rpm. The resulting complex/conjugate was further analyzed bySDS-PAGE and subsequent detection of Cy5-related fluorescence in thepolyacrylamide-gel. 15 μg of the complex/conjugate were mixed with 4×LDSsample buffer (Invitrogen) and incubated at 95° C. for 5 mM Cy5-relatedfluorescence was recorded using a LumiImager F1 device (Roche) at anexcitation wavelength of 645 nm.

The non-reduced samples show covalent site-specific disulfide bondformation for humanized anti-biotin antibody VH53C (FIG. 36, lane 4)with very low background fluorescence signal when humanized anti-biotinantibody without a cysteine in CDR2 was used (FIG. 36, lane 3).Biotin-Cys-Cy5 was also covalently coupled to humanized anti-digoxigeninantibody VH52bC (FIG. 36, lane 2) with a low background signal whenhumanized anti-digoxigenin antibody was used (FIG. 36, lane 1), but withsignificantly lower efficiency. This can be deduced from the excessBiotin-Cys-Cy5 that is detected on the bottom of the gel (arrows). Inthe case of humanized anti-digoxigenin antibody VH52bC significantlymore uncoupled Biotin-Cys-Cy5 can be detected (lane 2) than withhumanized anti-biotin antibody VH53C (lane 4). Upon reduction of thesamples, no Cy5-related fluorescence is detectable near the antibodyheavy- and light-chains, indicating that the covalent linkage was indeedformed by a disulfide bridge. Coomassie stains of each gel show that thetotal amount of protein in each lane was equal.

Example 20 Hapten-Directed Binding is a Prerequisite for the EfficientCovalent Coupling of Haptenylated Dyes or Polypeptides with anAnti-Hapten Cysteinylated Antibody

To show that affinity-driven complexation of haptenylated compounds withanti-hapten antibodies is a prerequisite for efficient disulfide-bondformation, the non-haptenylated peptide Ac-PYY(PEG3-Cys-4Abu-NH2)(Biosynthan 1763.1, SEQ ID NO: 178) was incubated with humanizedanti-digoxigenin antibody VH52bC and humanized anti-digoxigeninantibody. 1.4 mg of Ac-PYY(PEG3-Cys-4Abu-NH2) were dissolved in 100% DMFto a concentration of 10 mg/ml. 2 mg of each antibody was used in aconcentration of 11-13 mg/ml (about 75-89 μM) in a buffer composed of 50mM Tris-HCl, 1 mM EDTA, pH 8.2. Ac-PYY(PEG3-Cys-4Abu-NH2) and antibodieswere mixed at a 2.1:1 molar ratio (Ac-PYY(PEG3-Cys-4Abu-NH2 toantibody)). The peptide was added in 3 portions while the solution wasstirred at 500 rpm with a stirrer bar. Between each addition, sampleswere incubated for 5 mM at 200 rpm. After addition of the last portion,samples were incubated for 1 h at RT and 200 rpm.

The resulting complex/conjugate was defined as 97% monomeric IgG-likemolecule and 3% dimeric soluble aggregates for theAc-PYY(PEG3-Cys-4Abu-NH2): humanized anti-digoxigenin antibody VH52bCconjugate and as 100% monomeric for the Ac-PYY(PEG3-Cys-4Abu-NH2):humanized anti-digoxigenin antibody complex via size exclusionchromatography. Furthermore, the resulting complex/conjugate wasanalyzed by mass spectrometry. For the Ac-PYY(PEG3-Cys-4Abu-NH2):humanized anti-digoxigenin antibody VH52bC conjugate 17% of the detectedspecies was identified as antibody coupled to 2 peptide molecules, 51%was identified as antibody coupled to 1 peptide molecule and 32% wasidentified as antibody without coupled peptide. For theAc-PYY(PEG3-Cys-4Abu-NH2): humanized anti-digoxigenin antibody complex100% of the antibody was uncoupled.

Example 21 Disulfide Patterns that are Required for Formation ofProperly Folded Functional Hapten-Binding Antibodies with a CysteineMutation for Covalent Payload Coupling

Hapten-binding modules for covalent compound/payload coupling may becomposed of ‘standard’ antibodies such as IgGs which contain extracysteines that enable covalent attachment of haptenylatedcompounds/payloads. The method as reported herein introduces therequired functionalities (cysteines) within folded domains, whosestructure and sequence provide the basis for antibody functionality.Correct formation of defined disulfide bonds within as well as betweenthe domains of antibodies is essential for the formation and maintenanceof the correct structure and functionality. FIG. 37(A) shows thedisulfide pattern that is required to form functional binding arms suchas Fabs of unmodified antibodies, and FIG. 37(B) shows the disulfidepattern which is necessary to maintain structure and functionality ofthe VH52cB/VH53C mutated antibody derivative. To maintain the properdisulfide pattern, the additional cysteine that was introduced in the VHdomain must be unoccupied and must not interfere or react withneighboring cysteines. FIGS. 37(C) and 37(D) show that the additions ofthe extra cysteines generate possibilities to form incorrect disulfideswithin the VH domains during the biosynthesis of such molecules. Thefact that the VH52bC/VH53C position is located within the VH domain (andquite close to other cysteines) aggravates the risk that incorrectdisulfides may be formed during the biosynthesis of the heavy chain.Another potential problem is that VH and VL domains become assembledwithin the secretory pathway to one Fv fragment. The secretory pathwayinvolves redox-shuffling conditions and disulfide forming and—shufflingenzymes. Therefore, the potential to introduce incorrect disulfides byaddition of the VH52bC/VH53C mutation may ‘spread’ also to disulfides ofthe light chain (exemplarily shown in FIG. 37(E). This does furtherenhance the risk to obtain/generate improperly folded non-functionalmolecules. It is therefore quite surprising that—despite of theserisks—good amounts of homogeneous functional antibody derivatives thatcontain the VH52bC/VH53C mutation could be expressed and obtained, andwhich are capable to covalently connect to haptenylatedcompounds/payloads.

Example 22 Composition and Generation of Anti-HaptenDisulfide-Stabilized Single-Chain Fv Fragments with a Cysteine Mutationfor Covalent Coupling

Hapten-binding modules for covalent compound/payload coupling canconsist of ‘standard’ antibodies such as IgGs. Alternatively, they maybe modified entities such as recombinant Fv or Fab fragments, orderivatives thereof. Single-chain Fv fragments are frequently applied asalternative to full lengths antibodies, especially in applications wheresmall module size is required, or where additional binding modules aredesired to generate bi- or multispecific antibody derivatives. Oneexample for anti-hapten Fv-derived entities that have been generated isa disulfide-stabilized single-chain Fv which bind to and covalentlyconnects digoxigenylated compounds/payloads. The disulfide-stabilizedsingle-chain Fv with Dig-binding specificity was generated by connectinganti-digoxigenin antibody VH and VL domains via a flexible Gly and Serrich linker to each other. These VH and VL domains harbored in additioncysteine mutations in positions 44 of VH and position 100 of VL(positions according to Kabat et al.). These additional cysteines form astable intermolecular disulfide bond between VH and VL. This stabilizesthe scFv, as previously described (e.g. Reiter, Y., et al., NatureBiotechnology 14 (1996) 1239-1245).

In addition to that, another cysteine was introduced into the VH atposition 52b or 53, respectively, according to the Kabat numbering toadd the covalent linkage functionality to the Fv fragment.

However, introducing such a mutation into disulfide-stabilized Fvfragments is far more challenging than placing them into full lengthantibodies. Single-chain Fv fragments are inherently less stable thanfull length IgGs or Fab fragments because they lack constant domains asstabilizing and heterodimerization forcing entities. Stability can beconferred by placing additional cysteine mutations into the Fvs such asthe VH44-VL100 disulfide. However, this stabilizing principle works onlyif the disulfide forms at the correct positions between correctcysteines. Thus, in addition to defined intradomain disulfides (one inVH and one in VL), one single defined correct interdomain disulfideneeds to be formed. Disulfide connections between non-matching cysteineswill generate misfolded instable and non-functional entities.Considering that a disulfide-stabilized Fv fragment contains 6cysteines, 21 different disulfide connections can theoretically beformed—but only the right combination of 3 defined disulfides will forma functional stabilized scdsFv. This challenge is aggravated uponaddition of another free cysteine into the VH domain. The stabilizeddsFv that is desired contains two defined intradomain disulfides (oneeach in VH and VL), one defined interdomain disulfide (between VH andVL), and furthermore one free cysteine for haptenylated compound/payloadcoupling (in VH at position 52b/53). Considering that adisulfide-stabilized Fv fragment with extra cysteine mutation forcovalent coupling contains 7 cysteines, many different disulfideconnections can theoretically be formed but only the right combinationof the 3 defined disulfides, with the exact free cysteine position atVH52b/VH53 will result in a functional stabilized covalent couplingcompetent dsFv. One additional challenge is the fact that the additionalfree cysteine (VH52b/VH53) is located in close proximity to the VH44cysteine which is not a naturally occurring cysteine but a mutationintroduced for disulfide stabilization. VH44C is necessary for formingthe correct inter-domain disulfide, and this disulfide most likelywithout being bound by this theory forms after independent folding andassembly of VH and VL. Proximity of VH44C and VH52bC/VH53C aggravatesthe risk that the intradomain disulfide does not form in a correctmanner. But it has been found that functional disulfide stabilizedsingle-chain Fv modules that bind digoxigenin and that aresimultaneously capable to covalently connect to digoxigenylated payloadscan be produced. The composition of the disulfide-stabilizedsingle-chain Fv molecule that contains the correct disulfides and thefree cysteine in the correct position and its comparison to theundesired incorrectly folded molecules is shown in FIG. 38. Thesequences that encode the light chain variable regions and the modifiedheavy chain variable regions of this Dig-binding scdsFv with the VH52bCmutation Fv antibody derivative are listed under SEQ ID NO: 190 (VH) andthe corresponding VL under SEQ ID NO: 189. The successful generation ofsuch dsFv as modules for the generation of bispecific antibodyderivatives is described in the Example 23 (below), as well as in FIG.40(A), FIG. 40(B), and FIG. 40(C).

Example 23 Composition, Expression and Purification of BispecificAnti-Hapten Antibody Derivatives for Targeted Delivery of CovalentlyCoupled Compounds/Payloads

Bispecific antibodies were generated that contain hapten-bindingantibody modules for covalent compound/payload coupling. Theseantibodies additionally contain binding modules that enable targeting toother antigens. Applications for such bispecific antibodies includespecific targeting of haptenylated compounds/payloads to cells ortissues that carry the targeting antigen. One example for such moleculesthat was generated is a bispecific antibody with binding regions thatrecognize the tumor associated carbohydrate antigen LeY, andsimultaneously with disulfide-stabilized Fvs which bind and covalentlyconnect digoxigenylated compounds/payloads. Therefore,disulfide-stabilized single-chain Fvs were connected via flexible Glyand Ser rich connector peptides to the C-termini of the CH3 domains of aLeY antibody, resulting in tetravalent molecules with two LeY bindingarms and additionally two digoxigenin binding entities. Thedigoxigenin-binding entities harbored a VH44-VL100 disulfide bond whichhas been previously described (e.g. Reiter, Y., et al., NatureBiotechnology 14 (1996) 1239-1245). The digoxigenin binding entitycontained in addition the VH52bC mutation for covalent coupling. Thesequences that encode the light chain and the modified heavy chain ofthis LeY-Dig antibody derivative are listed under SEQ ID NO: 191 and SEQID NO: 192. The composition of the LeY-Dig bispecific antibodyderivative as delivery vehicle for covalently coupled compounds/payloadsis shown schematically in FIG. 39.

The bispecific molecules were generated by molecular biology techniques,expressed by secretion from cultured cells, subsequently purified fromculture supernatants in the same manner as described above. FIG. 40(A)shows the presence of modified H-chain and L-chain of this LeY-Dig(52bC) bispecific antibody in cell culture supernatants, visualized inWestern Blot analyses that detect antibody L-chains and H chains. FIG.40(B) demonstrates the homogeneity of these antibodies afterpurification by SDS-PAGE in the presence of a reducing agent. Stainingof the SDS-PAGE with Coomassie brilliant blue visualizes polypeptidechains related to the IgG with the apparent molecular sizes analogous totheir calculated molecular weights. FIG. 40(C) shows the SEC profile ofthe LeY-Dig(52bC) bispecific antibody after Protein A purification,demonstrating homogeneity and lack of aggregates in the proteinpreparations. Thus, bispecific antibodies which contain targetingmodules as well as modules for covalent coupling of haptenylatedcompounds/payloads can be generated and purified to homogeneity.

Example 24 X-Ray Structure Determination of Murine Anti-BiotinAntibody-Fab-Fragments in Complex with Biocytinamid

The protein structure of murine anti-Biotin antibody Fab-fragment wasdetermined in complex with biocytinamid. Therefore, crystals of theFab-fragment were grown in 0.8 M Succinic Acid, followed by charging ofthe antibody crystals with Biocytidinamide (diluted to 10 mM workingconcentration in crystallization solution, applied to the crystals inthe crystallization droplet). Crystals were washed three times with 2 μlof 10 mM Biocytidinamide solution and were finally incubated for 16 hrs.with Biocytidinamide at 21° C., harvested with 15% Glycerol ascryoprotectant and flash frozen in liquid nitrogen. Processeddiffraction images yielded a protein structure at 2.5 Å resolution. Thestructure and charge composition of the biotin-binding variable regionis shown in FIG. 46: Biotin binds into a surface pocket which is flankedby charged regions that composed of amino acids from the CDR regions.The complexed hapten is positioned in close proximity to a negativelycharged cluster of amino acids. Biotin which—as hapten—is derivatizedfor payload coupling at its carboxyl group binds with good efficacy asthere is no charge repulsion at this position (due to the lack of theCOOH group). In contrast, free (normal) biotin cannot bind efficient tothe antibody because its carboxyl group would be in close proximity tothis negative charge cluster, and hence becomes repulsed.

What is claimed is:
 1. An isolated antibody that binds to biotin,wherein the antibody comprises a heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 57; a heavy chain CDR2 comprising the aminoacid sequence of SEQ ID NO: 58; a heavy chain CDR3 comprising the aminoacid sequence of SEQ ID NO: 59; a light chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 61; a light chain CDR2 comprising the aminoacid sequence of SEQ ID NO:62; and a light chain CDR3 comprising theamino acid sequence of SEQ ID NO:
 63. 2. The antibody of claim 1,wherein the antibody comprises a variable heavy chain comprising theamino acid sequence of SEQ ID NO: 60 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:
 64. 3. An isolatedantibody that binds to theophylline, wherein the antibody comprises aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 89; aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 90; aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 91; alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 93; alight chain CDR2 comprising the amino acid sequence of SEQ ID NO:94; anda light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 95.4. The antibody of claim 3, wherein the antibody comprises a variableheavy chain comprising the amino acid sequence of SEQ ID NO: 92 and avariable light chain comprising the amino acid sequence of SEQ ID NO:96.
 5. An isolated antibody that binds to fluorescein, wherein theantibody comprises a heavy chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 105; a heavy chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 106; a heavy chain CDR3 comprising the amino acid sequenceof SEQ ID NO: 107; a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 109; a light chain CDR2 comprising the amino acid sequenceof SEQ ID NO:110; and a light chain CDR3 comprising the amino acidsequence of SEQ ID NO:
 111. 6. The antibody of claim 5, wherein theantibody comprises a variable heavy chain comprising the amino acidsequence of SEQ ID NO: 108 and a variable light chain comprising theamino acid sequence of SEQ ID NO:
 112. 7. An isolated antibody thatbinds to digoxigenin, wherein the antibody comprises a heavy chain CDR1comprising the amino acid sequence of SEQ ID NO: 25; a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 26; a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 27; a light chain CDR1comprising the amino acid sequence of SEQ ID NO: 29; a light chain CDR2comprising the amino acid sequence of SEQ ID NO:30; and a light chainCDR3 comprising the amino acid sequence of SEQ ID NO:
 31. 8. Theantibody of claim 7, wherein the antibody comprises a variable heavychain comprising the amino acid sequence of SEQ ID NO: 28 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:
 32. 9. Theantibody of claim 1, wherein the antibody is a multispecific antibodycomprising a first binding site that binds to biotin and a secondbinding site that specifically binds to a cell surface marker.
 10. Theantibody of claim 1, wherein the antibody is humanized.
 11. The antibodyof claim 3, wherein the antibody is a multispecific antibody comprisinga first binding site that binds to theophylline and a second bindingsite that specifically binds to a cell surface marker.
 12. The antibodyof claim 3, wherein the antibody is humanized.
 13. The antibody of claim5, wherein the antibody is a multispecific antibody comprising a firstbinding site that binds to fluorescein and a second binding site thatspecifically binds to a cell surface marker.
 14. The antibody of claim5, wherein the antibody is humanized.
 15. The antibody of claim 7,wherein the antibody is a multispecific antibody comprising a firstbinding site that binds to digoxigenin and a second binding site thatspecifically binds to a cell surface marker.
 16. The antibody of claim7, wherein the antibody is humanized.